Methods for treatment using anti-NMU agents

ABSTRACT

The inventors have produced two high specificity and high affinity monoclonal antibodies that bind to human neuromedin U (NMU). Methods and compositions are provided for treating an individual in need thereof (e.g., an individual who is obese and/or has diabetes) by administering an anti-NMU/NMUR agent (e.g., an anti-NMU antibody). For example, methods and compositions are provided for increasing circulating insulin in an individual. Methods and compositions are also provided for detecting neuromedin U (NMU) (e.g., in a biological sample such as serum). Methods and compositions are also provided for predicting whether an individual will develop diabetes and/or PDAC, and for identifying an individual who would benefit from administration of an anti-NMU/NMUR agent.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/288,985, filed Jan. 29, 2016, which is incorporatedherein by reference in its entirety

INTRODUCTION

The coupling of hormonal responses to nutrient availability isfundamental for metabolic control. In mammals, regulated secretion ofinsulin from pancreatic beta cells is a principal hormonal responseorchestrating metabolic homeostasis. Circulating insulin levelsconstitute a dynamic metabolic switch, signaling the fed state andnutrient storage (anabolic pathways) when elevated, or starvation andnutrient mobilization (catabolic pathways) when decreased.

Thus, insulin secretion must be precisely tuned to the nutritional stateof the animal. Increased circulating glucose stimulates beta celldepolarization and insulin secretion. In concert with glucose,gut-derived incretin hormones amplify glucose-stimulated insulinsecretion (GSIS) in response to ingested carbohydrates, thereby tuninginsulin output to the feeding state of the host.

There is a well-recognized ‘risk heterogeneity’ for morbidity andmortality in common human insulin-linked diseases like type 2 diabetes,obesity, pancreas cancer and pancreatitis. The combined burden of thesediseases on human health and economy is enormous.

There is a need in the art for new treatments (e.g., treatments thatregulate insulin secretion) and prediction methods for diseases such asdiabetes, obesity, pancreatic cancer, and pancreatitis.

SUMMARY

The inventors have shown that NMU is a hormone that regulates(suppresses) human insulin output by acting through its receptor,neuromedin U receptor (NMUR1), expressed on pancreatic beta-cells. Theinventors have generated and characterized mice lacking the NMUR1receptor, and have also produced two high specificity and high affinitymonoclonal antibodies (C578 and 2A16) that bind to human neuromedin U(NMU). These antibodies are injectable, block the interaction betweencirculating NMU and NMUR1 expressed on cells, and in vivo studies showthat injection of the antibodies is sufficient to suppress NMU levels,leading to enhanced insulin output and glucose handling. The inventorshave also shown that the antibodies can also be used as part of atwo-way enzyme linked immunosorption assay (ELISA) that is sensitiveenough to detect NMU in serum from mice as well as humans.

Measuring NMU (e.g., using a subject two-way ELISA) can effectivelystratify and identify broad subsets of patients with excessive NMUsignaling that may benefit from NMU antibody-based therapies. Forexample, using the two-way ELISA, NMU levels were shown to be elevatedin obesity states.

Methods and compositions are provided for treating an individual in needthereof (e.g., an individual who is obese and/or has diabetes, e.g.,type 2 diabetes) by administering an anti-NMU/NMUR agent (e.g., ananti-NMU antibody). For example, methods and compositions are providedfor increasing circulating insulin in an individual. Methods andcompositions are also provided for detecting neuromedin U (NMU) (e.g.,in a biological sample such as serum), e.g., using a two-way ELISA thatincludes one or two anti-NMU antibodies (or binding fragments thereof),each having the light chain and/or heavy chain CDRs of a new antibodydisclosed herein. Methods and compositions are also provided forpredicting whether an individual will develop diabetes, e.g., type 2diabetes, and/or PDAC, and for identifying an individual who wouldbenefit from administration of an anti-NMU/NMUR agent (e.g., in somecases using a subject two-way ELISA).

Provided are methods of treating an individual in need thereof (e.g., anindividual with diabetes, e.g., type 2 diabetes, or who is suspected ofhaving an increased risk of developing diabetes, e.g., type 2 diabetes;an individual who is obese and/or has a family history that includesdiabetics; an individual that has cystic fibrosis, familialpancreatitis, idiopathic pancreatitis, type 3c diabetes mellitus, latestage pancreatic cancer, and/or cancer cachexia; an individual who hasan increased level of circulating NMU relative to a reference level; andthe like), where the method includes administering to an individual, ata dose effective to increase the amount of circulating insulin in theindividual, an anti-NMU/NMUR agent that reduces binding between NMU andNMUR1. Also provided are methods of increasing circulating insulin in anindividual (e.g., an individual with diabetes, e.g., type 2 diabetes, orwho is suspected of having an increased risk of developing diabetes; anindividual who is obese and/or has a family history that includesdiabetics; an individual that has cystic fibrosis, familialpancreatitis, idiopathic pancreatitis, type 3c diabetes mellitus, latestage pancreatic cancer, and/or cancer cachexia; an individual who hasan increased level of circulating NMU relative to a reference level; andthe like), where the method includes administering to an individual, ata dose effective to increase the amount of circulating insulin in theindividual, an anti-NMU/NMUR agent that reduces binding between NMU andNMUR1.

With regard to either of the above methods, in some cases, theanti-NMU/NMUR agent binds to NMU. In some cases, the anti-NMU/NMUR agentis an anti-NMU antibody or antigen binding region thereof. In somecases, the anti-NMU antibody or antigen binding region thereof ishumanized. In some cases, the anti-NMU antibody or antigen bindingregion thereof comprises a light chain comprising a CDR-L1, CDR-L2, andCDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs:18-20, respectively, or SEQ ID NOs: 2-4, respectively. In some cases,the anti-NMU antibody or antigen binding region thereof comprises aheavy chain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the aminoacid sequences set forth in SEQ ID NOs: 26-28, respectively, or SEQ IDNOs: 10-12, respectively. In some cases, the anti-NMU antibody orantigen binding region thereof comprises a light chain comprising theamino acid sequence set forth in any one of SEQ ID NOs: 17, 38, 1, and36. In some cases, the anti-NMU antibody or antigen binding regionthereof comprises a heavy chain comprising the amino acid sequence setforth in any one of SEQ ID NOs: 25, 39, 9, and 37. In some cases, theanti-NMU antibody or antigen binding region thereof comprises a lightchain comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 18-20, respectively, and a heavychain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 26-28. In some cases, the anti-NMUantibody or antigen binding region thereof comprises a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 2-4, respectively, and a heavy chaincomprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12.

Also provided are proteins that bind specifically to NMU and include anantigen binding region that includes one or more of: (a) a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 18-20, respectively, or SEQ ID NOs:2-4, respectively; and (b) a heavy chain comprising a CDR-H1, CDR-H2,and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs:26-28, respectively, or SEQ ID NOs: 10-12, respectively. In some cases,the antigen binding region comprises one or more of: (a) a light chaincomprising the amino acid sequence set forth in any one of SEQ ID NOs:17, 38, 1, and 36; and (b) a heavy chain comprising the amino acidsequence set forth in any one of SEQ ID NOs: 25, 39, 9, and 37. In somecases, the antigen binding region comprises a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 18-20, respectively, and a heavy chain comprising aCDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forthin SEQ ID NOs: 26-28, respectively. In some cases, the antigen bindingregion comprises a light chain that comprises the amino acid sequenceset forth in any one of SEQ ID NOs: 17 and 38, and a heavy chain thatcomprises the amino acid sequence set forth in any one of SEQ ID NOs: 25and 39. In some cases, the antigen binding region comprises a lightchain comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 2-4, respectively, and a heavy chaincomprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively. In some cases,the antigen binding region comprises a light chain that comprises theamino acid sequence set forth in any one of SEQ ID NOs: 1 and 36, and aheavy chain that comprises the amino acid sequence set forth in any oneof SEQ ID NOs: 9 and 37. In some cases, the protein is an antibody(e.g., a mouse or human antibody). In some cases, the antibody is ahumanized antibody. Also provided are nucleic acids (e.g., expressionvectors) encoding the above proteins. Also provided are cells thatinclude one or more nucleic acids (e.g., expression vectors) encodingthe above proteins.

Provided are kits (e.g., for detecting neuromedin U (NMU)) the include afirst anti-NMU antibody and a second anti-NMU antibody, where the firstand second anti-NMU antibodies bind to non-overlapping amino acids ofNMU, and where one of the first and second anti-NMU antibody antibodiesincludes: (a) a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 18-20,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 26-28,respectively; or (b) a light chain comprising a CDR-L1, CDR-L2, andCDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 2-4,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 10-12,respectively. In some cases: (i) one of the first and second anti-NMUantibodies comprises a light chain comprising a CDR-L1, CDR-L2, andCDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs:18-20, respectively, and a heavy chain comprising a CDR-H1, CDR-H2, andCDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs:26-28, respectively; and (ii) the other of the first and second anti-NMUantibodies comprises a light chain comprising a CDR-L1, CDR-L2, andCDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 2-4,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 10-12,respectively. In some cases, the first anti-NMU antibody is immobilizedon a solid surface. In some cases, the solid surface is a bead or asurface of a well of a multi-well plate. In some cases, the secondanti-NMU antibody is detectably labeled. In some cases, the secondanti-NMU antibody is conjugated to a fluorophore, a fluorescent protein,or an enzyme that is indirectly detectable.

Provided are methods for detecting neuromedin U (NMU), where the methodsinclude measuring an amount of NMU in a sample using a subject kit(e.g., any of the kits described above), where the method includescontacting NMU in the sample with the first and second anti-NMUantibodies, and measuring an amount of the second anti-NMU antibody. Insome cases, the method includes: (a) contacting the sample with thefirst anti-NMU antibody, wherein the first anti-NMU antibody isimmobilized on a solid surface and NMU of the sample binds to the firstanti-NMU antibody; (b) contacting the NMU that is bound to the firstanti-NMU antibody with the second anti-NMU antibody; and (c) measuringan amount of the second anti-NMU antibody. In some cases, the methodincludes performing a first wash step between steps (a) and (b), andperforming a second wash step between steps (b) and (c).

Provided are methods of predicting whether an individual will developdiabetes, e.g., type 2 diabetes, where the methods include: (a)measuring an amount of NMU present in a blood sample from an individual,(b) determining that the amount of NMU present in the blood sample isgreater than or equal to a reference value; and (c) predicting that theindividual will develop diabetes, e.g., type 2 diabetes. In some cases,the individual is suspected of having an increased risk of developingdiabetes, e.g., type 2 diabetes. In some cases, the individual has afamily history of diabetes, e.g., type 2 diabetes. In some cases, theindividual is overweight. In some cases, the individual is obese.

Provided are methods of predicting whether an individual will developPDAC or pancreatitis, where the methods include: (a) measuring anexpression level of NMU in a biological sample from an individual, (b)determining that the measured expression level of NMU is greater than orequal to a reference value; and (c) predicting that the individual willdevelop PDAC or pancreatitis. In some cases, the individual is suspectedof having an increased risk of developing PDAC or pancreatitis. In somecases, the individual has a family history of PDAC or pancreatitis.

Provided are methods of identifying an individual who would benefit fromadministration of an anti-NMU/NMUR agent, where the methods include: (a)measuring an amount of NMU present in a blood sample from an individual,(b) determining that the amount of NMU present in the blood sample isgreater than or equal to a reference value; and (c) predicting that theindividual would benefit from administration of an anti-NMU/NMUR agent.In some cases, the individual is suspected of having an increased riskof developing diabetes, e.g., type 2 diabetes, PDAC, or pancreatitis. Insome cases, the individual has a family history of diabetes, e.g., type2 diabetes, PDAC, or pancreatitis. In some cases, the individual isoverweight. In some cases, the individual is obese.

In some cases (e.g., in any of the above methods), the sample is a bloodsample. In some cases, the blood sample is a sample collected after anovernight fast. In some cases, the blood sample is a sample collectedafter a provocative challenge (e.g., a glucose challenge). In somecases, the measuring is not performed on exosomes isolated from theblood sample. In some cases, the measuring includes use of a two-wayELISA. In some cases, the measuring comprises contacting the NMU with aprotein that: (i) binds specifically to NMU and (ii) includes an antigenbinding region that comprises one or more of: (a) a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 18-20, respectively, or SEQ ID NOs:2-4, respectively; and (b) a heavy chain comprising a CDR-H1, CDR-H2,and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs:26-28, respectively, or SEQ ID NOs: 10-12, respectively. In some cases,the antigen binding region comprises one or more of: (a) a light chaincomprising the amino acid sequence set forth in any one of SEQ ID NOs:17, 38, 1, and 36; and (b) a heavy chain comprising the amino acidsequence set forth in any one of SEQ ID NOs: 25, 39, 9, and 37. In somecases, the antigen binding region comprises a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 18-20, respectively, and a heavy chain comprising aCDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forthin SEQ ID NOs: 26-28, respectively. In some cases, the antigen bindingregion comprises a light chain that comprises the amino acid sequenceset forth in any one of SEQ ID NOs: 17 and 38, and a heavy chain thatcomprises the amino acid sequence set forth in any one of SEQ ID NOs: 25and 39. In some cases, the antigen binding region comprises a lightchain comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 2-4, respectively, and a heavy chaincomprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively. In some cases,the antigen binding region comprises a light chain that comprises theamino acid sequence set forth in any one of SEQ ID NOs: 1 and 36, and aheavy chain that comprises the amino acid sequence set forth in any oneof SEQ ID NOs: 9 and 37. In some cases, the protein is an antibody. Insome cases, the antibody is a mouse or human antibody. In some cases,the antibody is a humanized antibody. In some cases, the method includesa step of collecting the blood sample (e.g., prior to the measuringstep, prior to contacting the sample with an anti-NMU/NMUR agent, andthe like).

In some cases, a subject method (e.g., a diagnostic method, a method ofpredicting, a method of identifying) includes (e.g., after a step ofpredicting), administering to the individual an anti-NMU/NMUR agent thatreduces interaction between NMU and NMUR1 (e.g., an anti-NMU antibody),at a dose effective to increase the amount of circulating insulin in theindividual.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1E. Depict data related to physiological dynamics of human andmouse NMU in vivo revealed by NMU ELISA assays.

FIG. 2A-2F. Depict data related to NMU suppressing post-prandial outputin humans and mice.

FIG. 3A-3F. Depicts data related to in vivo loss of NMUR1 signalingpathway improving glucose clearance during prolonged fasting.

FIG. 4A-4C. Depicts data related to NMU dynamics in the pathologicalsetting of obesity.

FIG. 5A-5C. Depicts data related to NMU signaling loss in vivo improvingmetabolism in obesity.

FIG. 6A-6E. Depicts data related to NMU as a novel regulator of GLP1 andGIP.

FIG. 7A-7D. Depicts data related to NMU signaling attenuation enhancingincretin output.

FIG. 8A-8B. Depicts data related to NMU signaling decreasing insulin andincreasing glucagon output in vivo.

FIG. 9A-9B. Depicts data related to anti-NMU antibody therapy promotinginsulin secretion and eliminating NMU-induced glucose intolerance.

FIG. 10. Depicts CDR sequences obtained from both light and heavy chainsof newly generated anti-NMU antibodies (2A16 and C578).

FIG. 11A-11B. Depicts NMU immunoreactivity. vl=mucosal villus, gl=gland

FIG. 12A-12C. FIG. 12A depicts data related to NmuR1 production beingrestricted to insulin⁺β cells. FIG. 12B-12C depict data related toshowing that human NMU potently suppressed glucose-stimulated insulinsecretion from human islets,

FIG. 13A-13F. FIG. 13A depicts data showing that Nmu infusion led to atwo-fold increase of mean serum Nmu levels. FIG. 13B-13D depicts datarelated to serum insulin and GLP-1 levels, which were reduces, and toglucagon levels, which were increased. FIG. 13E-13F depict data showingthat reduced insulin secretion and impaired glucose tolerance weredetected after oral glucose tolerance testing in mice infused with Nmu.

FIG. 14A-140. Depict data related to NMU mis-expression in in chronicpancreatitis or pancreatic ductal adenocarcinoma.

FIG. 15. Depicts data related to co-labelling with antibodies specificfor NMU, Insulin and Glucagon, which demonstrated that Glucagon⁺ cells(but not Insulin⁺ cells) mis-expressed NMU.

FIG. 16. Depicts data related to levels of mRNA encoding NMU in patientswith PDAC. Depicted results are of a meta-analysis: NMU mRNA expressionis increased in PDAC. The x-axis represents the standardized meandifference, effect size from each dataset, between PDAC and normal inlog 2 scale. The size of the square is proportional to weighted inverseof the variance, of each study-specific effect size. The whiskers arethe 95% confidence interval of the effect size of each study. The yellowdiamond and its width represent the pooled effect size and the 95%confidence interval for a given gene, respectively.

FIG. 17. Depicts data related to measurements of serum NMU using asubject two-way ELISA. NMU was measured in serum from control subjectswithout cancer or from subjects with advanced pancreatic ductaladenocarcinoma (PDAC).

FIG. 18. Depicts serum levels of NMU measured from subjects.

FIG. 19A-19B. Depict schematic representations of NMU signaling innormal and diseased states.

FIG. 20A-20C. Depict micrographs and schematic representations of NMUexpression in humans and mice, and depicts data from RNA-Seq analysis ofmouse duodenal and ileal Nmu-eGFP⁺ cells.

FIG. 21A-21C. Depict data showing NMUR1 expression in human β-cells andshowing that NMU suppresses insulin secretion.

FIG. 22A-22C. Depict data showing serum NMU levels in mice and humans.

FIG. 23A-23F. Depict data related to in vivo NMU-23 infusion in mice.

FIG. 24A-24F. Depict data related to genetics with primary human isletcells (e.g., using lentiviral vectors to express transgenes), and assaysin pseudoislets.

FIG. 25. Depicts data showing that NMU suppression of insulin secretionby cultured mouse islets requires NmrR1.

FIG. 26. Depicts data showing that NMU fails to suppress stimulation ofinsulin secretion by pertussis toxin.

FIG. 27A-27D. Depicts data related to starvation diabetes and NMUinjection. FIG. 27A-28B relate to starvation diabetes in wildtype B6mice. FIG. 27C-27D relate to NMU injection reconstituting starvationdiabetes.

DETAILED DESCRIPTION

The inventors have shown that NMU is a hormone that regulates humaninsulin output by acting through its receptor, neuromedin U receptor(NMUR1), expressed on pancreatic beta-cells. The inventors havegenerated and characterized mice lacking the NMUR1 receptor, and havealso produced two high specificity and high affinity monoclonalantibodies (C578 and 2A16) that bind to human neuromedin U (NMU). Theseantibodies are injectable, block the interaction between circulating NMUand NMUR1 expressed on cells, and in vivo studies show that injection ofthe antibodies is sufficient to suppress NMU levels, leading to enhancedinsulin output and glucose handling. The inventors have also shown thatthe antibodies can also be used as part of a two-way enzyme linkedimmunosorption assay (ELISA) that is sensitive enough to detect NMU inserum from mice as well as humans.

Methods and compositions are provided for treating an individual in needthereof (e.g., an individual who is obese and/or has diabetes, e.g.,type 2 diabetes) by administering an anti-NMU/NMUR agent (e.g., ananti-NMU antibody). For example, methods and compositions are providedfor increasing circulating insulin in an individual. Methods andcompositions are also provided for detecting neuromedin U (NMU) (e.g.,in a biological sample such as serum). Methods and compositions are alsoprovided for predicting whether an individual will develop diabetes,e.g., type 2 diabetes, and/or Pancreatic Ductal Adenocarcinoma (PDAC),and for identifying an individual who would benefit from administrationof an anti-NMU/NMUR agent.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

In the description that follows, a number of terms conventionally usedin the field are utilized. In order to provide a clear and consistentunderstanding of the specification and claims, and the scope to be givento such terms, the following definitions are provided.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an alpha carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired (e.g., mice, non-humanprimates, humans, etc.). “Mammal” for purposes of treatment refers toany animal classified as a mammal, including humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, horses, cats,cows, sheep, goats, pigs, etc. In some cases, an individual of a subjectmethod is a mammal. In some embodiments, the mammal is a rodent (e.g., arat, a mouse), in some cases the mammal is a non-human primate, and insome cases the mammal is a human.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas cancer cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc.

The term “biological sample” encompasses a clinical sample such asblood, plasma, serum, aspirate, and also includes tissue obtained bysurgical resection, tissue obtained by biopsy, cells in culture, cellsupernatants, cell lysates, tissue samples, organs, bone marrow, and thelike. A “biological sample” includes biological fluids derived therefrom(e.g., cancerous cell, infected cell, etc.), e.g., a sample comprisingpolynucleotides and/or polypeptides that is obtained from such cells(e.g., a cell lysate or other cell extract comprising polynucleotidesand/or polypeptides). A biological sample comprising an inflicted cell(e.g., cancer cell, an infected cell, etc.) from a patient can alsoinclude non-inflicted cells. In some cases, a biological sample (e.g.,one in which an amount/concentration of NMU is measured) is serum. Insome such cases, NMU is measured in the serum and not from sub-fractionsof the serum. For example, in some cases, NMU is measured in a serumsample that includes exosomes (e.g., a serum sample without or prior toany sub-fractioning to isolate exosomes). In some cases, NMU is measuredin a serum sample that does not include exosomes (e.g., in some casesexosomes are removed from (separated from) the serum prior to measuringNMU in the serum).

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of cancer, the determination thatan individual is at risk for developing diabetes, e.g., type 2 diabetes,and the like.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of disease progression (e.g., progression to diabetes,progression to cancer, etc.), including recurrence, metastatic spread ofcancer, and drug resistance.

The term “prediction” is used herein to refer to the act of foretellingor estimating, based on observation, experience, or scientificreasoning. In one example, a physician may predict whether a patientwill become diabetic (e.g., whether an individual who is obese willbecome diabetic). As another example, one may predict the likelihoodthat an individual will progress to pancreatic cancer (e.g., predictingwhether an individual with pancreatitis will progress to pancreaticcancer, whether the individual will develop PDAC, etc.)

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides/epitopes). In some embodiments,the affinity of one molecule for another molecule to which itspecifically binds is characterized by a K_(D) (dissociation constant)of 10⁻⁵ M or less (e.g., 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less,10⁻⁹ M or less, 10⁻¹⁰ M or less, 10¹¹ M or less, 10⁻¹² M or less, 10⁻¹³M or less, 10⁻¹⁴ M or less, 10⁻¹⁶ M or less, or 10⁻¹⁶ M or less).“Affinity” refers to the strength of binding, increased binding affinitybeing correlated with a lower K_(D).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member). Examples of specificbinding members include, but are not limited to: agents thatspecifically bind NMU and/or NMUR1 (e.g., antibodies).

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments (e.g., Fab fragments) solong as they exhibit the desired biological activity. “Antibodies” (Abs)and “immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light- and heavy-chain variable domains (Clothia et al., J.Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.U.S.A. 82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the b-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity. The CDRs of the light chain arereferred to as CDR-L1, CDR-L2, and CDR-L3, while the CDRs of the heavychain are referred to as CDR-H1, CDR-H2, and CDR-H3.

Digestion of antibodies (e.g., with enzymes such as papain, Ficin, andthe like) produces two identical antigen-binding fragments, called “Fab”fragments, each with a single antigen-binding site, and a residual “Fc”fragment, whose name reflects its ability to crystallize readily. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species(scFv), one heavy- and one light-chain variable domain can be covalentlylinked by a flexible peptide linker such that the light and heavy chainscan associate in a “dimeric” structure analogous to that in a two-chainFv species. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

As would be readily understood by one of ordinary skill in the art, itis noted that designation of the terms “light chain” and “heavy chain”CDRs can be arbitrary, provided that the two halves of the binding sitehave matched sets of CDRs. As an example, in a scFv, CDR 1, 2, 3 can beon the heavy side or the light side, provided that it is matched withCDR 4, 5, 6 on the opposite chain. Thus, in cases where CDRs are statedto be heavy chain or light chain CDRs. Thus, when a subject anti-NMUantibody or antigen binding region thereof includes a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 18-20, respectively, and a heavychain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 26-28, respectively, it is also meantto encompass an anti-NMU antibody or antigen binding region thereof inwhich the heavy chain CDRs include the amino acid sequences set forth inSEQ ID NOs: 18-20 and the light chain CDRs include the amino acidsequences set forth in SEQ ID NOs: 26-28. Likewise, when a subjectanti-NMU antibody or antigen binding region thereof comprises a lightchain comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 2-4, respectively, and a heavy chaincomprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively, it is also meantto encompass an anti-NMU antibody or antigen binding region thereof inwhich the heavy chain CDRs include the amino acid sequences set forth inSEQ ID NOs: 2-4 and the light chain CDRs include the amino acidsequences set forth in SEQ ID NOs: 10-12.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called a, d, e, g, and m, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. Engineered variants of immunoglobulinsubclasses, including those that increase or decrease immune effectorfunctions, half-life, or serum-stability, are also encompassed by thisterminology.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

Unless specifically indicated to the contrary, the term “conjugate” asdescribed and claimed herein is defined as a heterogeneous moleculeformed by the covalent attachment of one or more antibody fragment(s) toone or more polymer molecule(s), where the heterogeneous molecule iswater soluble, i.e. soluble in physiological fluids such as blood, andwherein the heterogeneous molecule is free of any structured aggregate.A conjugate of interest is PEG. In the context of the foregoingdefinition, the term “structured aggregate” refers to (1) any aggregateof molecules in aqueous solution having a spheroid or spheroid shellstructure, such that the heterogeneous molecule is not in a micelle orother emulsion structure, and is not anchored to a lipid bilayer,vesicle or liposome; and (2) any aggregate of molecules in solid orinsolubilized form, such as a chromatography bead matrix, that does notrelease the heterogeneous molecule into solution upon contact with anaqueous phase. Accordingly, the term “conjugate” as defined hereinencompasses the aforementioned heterogeneous molecule in a precipitate,sediment, bioerodible matrix or other solid capable of releasing theheterogeneous molecule into aqueous solution upon hydration of thesolid.

As used in this disclosure, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly, e.g., to asubject anti-NMU/NMUR agent. The label may itself be detectable byitself (directly detectable label) (e.g., radioisotope labels orfluorescent labels), or the label can be indirectly detectable, e.g., inthe case of an enzymatic label, the enzyme may catalyze a chemicalalteration of a substrate compound or composition and the product of thereaction is detectable.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

Compositions

Provided are compositions and methods for treatment (e.g., forincreasing circulating insulin in an individual, for treating anindividual who is obese or who has or is at risk of developing diabetes,for treating an individual who has chronic pancreatitis, familialpancreatitis, idiopathic pancreatitis, type 3c diabetes mellitus, cysticfibrosis, late stage pancreatic cancer, and/or cancer cachexia), and forprediction (e.g., predicting whether an individual will developdiabetes, predicting whether an individual will develop PDAC, and/orpredicting whether an individual would benefit from administration of ananti-NMU/NMUR agent). A subject method of treatment includesadministration of an anti-NMU/NMUR agent (e.g., an anti-NMU antibody orantigen-binding fragment thereof) and a subject method of predictionincludes measuring an expression level of neuromedin U (NMU) (e.g.,measuring an amount of NMU protein present in a biological sample).

Provided are new anti-NMU antibodies, which are anti-NMU/NMUR agents(e.g., see the Examples below). Human NMU is a 25 amino acid hormoneencoded by the gene NMU and produced from a processed pre-prohormone(Mitchell et al., Br J Pharmacol. 2009 September; 158(1):87-103). NMU isproduced in the gastrointestinal tract of humans and other mammals likemice. NMU has no known covalent modifications other than C-terminalamidation (a common feature of circulating peptide hormones), and thenewly generated antibodies (C578 and 2A16) were generated against abioactive form of circulating NMU (FRVDEEFQSPFASQSRGYFLFRPRN-NH₂) (SEQID NO: 33).

The following amino acid sequences are those of pre-processed human andmouse neuromedin U (NMU) as downloaded from NCB:

Human NMU (isoform 1) (NP_006672.1) (SEQ ID NO: 40)MLRTESCRPRSPAGQVAAASPLLLLLLLLAWCAGACRGAPILPQGLQPEQQLQLWNEIDDTCSSFLSIDSQPQASNALEELCFMIMGMLPKPQEQDEKDNTKRFLFHYSKTQKLGKSNVVSSVVHPLLQLVPHLHERRMKR FRVDEEFQS PFASQSRGYFLFRPRNGRRSAGFI * amino acids of the processed form are bold and underlinedHuman NMU (isoform 2) (NP_001278974.1) (SEQ ID NO: 41)MLRTESCRPRSPAGQVAAASPLLLLLLLLAWCAGACRGAPILPQGLQPEQQLQLWNEASNALEELCFMIMGMLPKPQEQDEKDNTKRFLFHYSKTQKLGKSNVVSSVVHPLLQLVPHLHERRMKR FRVDEEFQSPFASQSRGYFLFRPRN GRRSAGFI* amino acids of the processed form are bold and underlinedMouse NMU (NP_062388.1) (SEQ ID NO: 42)MSRAAGHRPGLSAGQLAAATASPLLSLLLLLACCADACKGVPISPQRLQPEQELQLWNEIHEACASFLSIDSRPQASVALRELCRIVMEISQKPQEQSEKDNTKRFLFHYSKTQKLGNSNVVSSVVHPLLQLVPQLHERRMKRFKAEYQS PSVG QSKGYFLFRPRNGKRSTSFI * amino acids recognized by the antibody C578 arebold and underlinedAnti-NMU/NMUR Agent.

NMU (e.g., circulating NMU in the serum) binds to its receptor (NMUR1)on the surface of cells that express NMUR1. An “anti-NMU/NMUR agent”binds to (specifically binds) NMU and/or NMUR1 and inhibits/blocks theinteraction between (e.g., reduces the binding between) NMU and NMUR1.This binding leads to decreased NMU signaling.

In some case, an anti-NMU/NMUR agent is an anti-NMU antibody or bindingfragment thereof. The inventors have produced two new monoclonalanti-NMU antibodies (referred to herein as “C578” and “2A16”), both ofwhich specifically bind to human NMU (antibody C578 also specificallybinds to mouse NMU, i.e., it cross-reacts). Sequence details, includingthe CDR sequences, and additional information related to the antibodiesis presented in FIG. 10.

As such, in some cases, a subject anti-NMU antibody (or antigen bindingfragment thereof) includes a light chain comprising a CDR-L1, CDR-L2,and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs:2-4, respectively. In some cases, a subject anti-NMU antibody (orantigen binding fragment thereof) includes a heavy chain comprising aCDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forthin SEQ ID NOs: 10-12, respectively. In some cases, a subject anti-NMUantibody (or antigen binding fragment thereof) includes a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 2-4, respectively, and a heavy chaincomprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively. In some cases, asubject anti-NMU antibody (or antigen binding fragment thereof) includesthe antigen binding region (e.g., the CDRs, and in some cases theframework region as well) of the C578 antibody. In some cases, a subjectanti-NMU antibody is the C578 antibody. In some cases, a subjectanti-NMU antibody is a humanized version of the C578 antibody (e.g., theantibody includes the CDRs of the C578 antibody but is humanized).

In some cases, a subject anti-NMU antibody (or antigen binding fragmentthereof) includes a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 18-20,respectively. In some cases, a subject anti-NMU antibody (or antigenbinding fragment thereof) includes a heavy chain comprising a CDR-H1,CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQID NOs: 26-28, respectively. In some cases, a subject anti-NMU antibody(or antigen binding fragment thereof) includes a light chain comprisinga CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences setforth in SEQ ID NOs: 18-20, respectively, and a heavy chain comprising aCDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forthin SEQ ID NOs: 26-28, respectively. In some cases, a subject anti-NMUantibody (or antigen binding fragment thereof) includes the antigenbinding region (e.g., the CDRs, and in some cases the framework regionas well) of the 2A16 antibody. In some cases, a subject anti-NMUantibody is the 2A16 antibody. In some cases, a subject anti-NMUantibody is a humanized version of the 2A16 antibody (e.g., the antibodyincludes the CDRs of the 2A16 antibody but is humanized).

The term “NMU-neutralizing antibody” is used herein to mean an antibody(or antigen binding fragment thereof) that reduces that activity of NMU(e.g., an antibody that binds to NMU and blocks the interaction betweenNMU and its receptor (NMUR1)).

Suitable anti-NMU antibodies (or anti-NMUR1 antibodies) include fullyhuman, humanized or chimeric versions of such antibodies. For example,humanized antibodies are useful for in vivo applications in humans dueto their low antigenicity. Similarly caninized, felinized, etc.antibodies are useful for applications in dogs, cats, and other speciesrespectively. Antibodies of interest include humanized antibodies, orcaninized, felinized, equinized, bovinized, porcinized, etc.,antibodies, and variants thereof. Also envisioned are single chainantibodies derived from camelids, single chain antibodies derived fromshark, engineered fibronectin domain-containing proteins, knottinpeptides, and DARPins; and fluorophore-conjugated versions of each ofthese reagents.

In some cases, an anti-NMU antibody (e.g., one that includes the lightand heavy chain CDRs of C578 or 2A16) is a humanized antibody (e.g., canbe an IgG4 isotype humanized antibody, e.g., an IgG4 isotype antibodyhaving a mutation in the hinge region such as the S241P mutation thatreduces heterogeneity sometimes found in chimeric mouse/human IgG4antibodies)(e.g., see Angal et al., Mol Immunol. 1993 January;30(1):105-8). In other words, in some cases, an anti-NMU antibody (e.g.,one that includes a light chain with CDR-L1, CDR-L2, and CDR-L3 havingthe amino acid sequences set forth in SEQ ID NOs: 2-4, respectively, anda heavy chain with CDR-H1, CDR-H2, and CDR-H3 having the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively; or one thatincludes a light chain with CDR-L1, CDR-L2, and CDR-L3 having the aminoacid sequences set forth in SEQ ID NOs: 18-20, respectively, and a heavychain with CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequencesset forth in SEQ ID NOs: 26-28, respectively) is a humanized antibody(e.g., can be an IgG4 isotype humanized antibody, e.g., an IgG4 isotypeantibody having a mutation in the hinge region such as the S241Pmutation that reduces heterogeneity sometimes found in chimericmouse/human IgG4 antibodies).

In general, humanized antibodies are made by substituting amino acids inthe framework regions of a parent non-human antibody to produce amodified antibody that is less immunogenic in a human than the parentnon-human antibody. For example, in some cases, antibody humanizationinvolves placing the complementarity determining regions (CDRs) into the‘framework’ of a human antibody, leading to production of a chimericantibody compatible with human in vivo use.

Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chainshuffling (U.S. Pat. No. 5,565,332). In certain embodiments, frameworksubstitutions are identified by modeling of the interactions of the CDRand framework residues to identify framework residues important forantigen binding and sequence comparison to identify unusual frameworkresidues at particular positions (see, e.g., U.S. Pat. No. 5,585,089;Riechmann et al., Nature 332:323 (1988)). Additional methods forhumanizing antibodies contemplated herein are described in U.S. Pat.Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493;5,558,864; 4,935,496; and 4,816,567, and PCT publications WO 98/45331and WO 98/45332.

In some embodiments, therefore, the disclosure provides humanizedversions of the above described monoclonal antibodies (e.g., thoseantibodies that recognize human NMU). For any of the described anti-NMUantibodies, the antibody can be a humanized antibody, a binding fragmentthereof (e.g., a Fab fragment), or any permutation having the antigenbinding domain (or, e.g., the CDRs of the antigen binding domain).(e.g., see definition of “antibody” above).

In some case, an anti-NMU/NMUR agent is an RNAi agent (e.g., ananti-NMUR1 RNAi agent or an anti-NMU RNAi agent). For example, geneticstudies presented in the examples below demonstrate results in miceharboring genetic ablation of NMUR1. Such ablation can be temporarily orpermanently mimicked using RNAi agents, e.g., an RNAi agent that targets(e.g., is specific for) NMUR1, or an RNAi agent that targets (e.g., isspecific for) NMU.

The term “RNAi agent” is used herein to mean any agent that can be usedto induce a gene specific RNA interference (RNAi) response in a cell.Suitable examples of RNAi agents include, but are not limited to shortinterfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), and microRNAs (miRNA). An RNAi agent (e.g., shRNA, siRNA, miRNA) specific for NMUis an agent that targets the mRNA encoding the NMU protein. An RNAiagent (e.g., shRNA, siRNA, miRNA) specific for NMUR1 is an agent thattargets the mRNA encoding the NMUR1 protein. RNAi agents can readily bedesigned to specifically target any desired mRNA (e.g., one encoding NMUor NMUR1) by choosing an appropriate nucleotide sequence.

Various RNAi agent designs (RNAi agents with various features) are knownin the art and any convenient RNAi agent (e.g., one that targets NMU orone that targets NMUR1) can be used. For example, various designs ofRNAi agents (as well as methods of their delivery) can be found innumerous patents, including, but not limited to U.S. Pat. Nos.7,022,828; 7,176,304; 7,592,324; 7,667,028; 7,718,625; 7,732,593;7,772,203; 7,781,414; 7,807,650; 7,879,813; 7,892,793; 7,910,722;7,947,658; 7,973,019; 7,973,155; 7,981,446; 7,993,925; 8,008,271;8,008,468; 8,017,759; 8,034,922; 8,399,653; 8,415,319; 8,426,675;8,466,274; 8,524,679; 8,524,679; 8,569,065; 8,569,256; 8,569,258;9,233,102; 9,233,170; and 9,233,174; all of which are incorporatedherein by reference.

Two-Way ELISA Assay (Sandwich ELISA)

Provided are compositions (e.g., kits) and methods for measuring NMU ina sample (e.g., in a blood sample, in a serum sample, in a plasmasample, and the like). In some cases, a subject method of detection is atwo-way enzyme-linked immunosorbent assay (two-way ELISA). In somecases, in a subject two-way ELISA, a first antibody is immobilized on asolid surface (e.g., a bead, the surface of a well in a multi-wellplate, etc.). The first antibody binds to an antigen (in this case NMU),and then a second antibody is used that also binds to the antigen, butbinds to a different region of the antigen than the region to which thefirst antibody binds. In other words, the first and second antibodiesbind to non-overlapping amino acids of NMU.

In some cases, a subject method is a method of detecting NMU, andincludes a step of measuring an amount of NMU present in a biologicalsample using a subject two-way ELISA (e.g., as described here and in thekits below). Such methods include contacting a biological sample (e.g.,contacting NMU in the biological sample) with the first and secondanti-NMU antibodies, and measuring an amount of the second anti-NMUantibody. For example, in some cases, the method includes (a) contactinga biological sample (e.g., serum sample) with a first anti-NMU antibody,wherein the first anti-NMU antibody is immobilized on a solid surfaceand NMU of the biological sample binds to the first anti-NMU antibody;(b) contacting the NMU that is bound to the first anti-NMU antibody,with the second anti-NMU antibody; and (c) measuring an amount of thesecond anti-NMU antibody. In some cases, the method includes a firstwash step between steps (a) and (b), and/or performing a second washstep between steps (b) and (c). In some cases the biological sample is ablood sample. In some cases the biological sample is a serum sample. Insome cases the biological sample is an aspirate. In some cases thebiological sample is a biopsy (e.g., from a biopsy).

In some cases, the second antibody is detectably labeled (e.g.,conjugated to an indirectly or directly detectable label). In somecases, the second antibody is not conjugated to a label but isnonetheless detectable (e.g., using secondary antibodies, e.g., if thesecond antibody is a mouse or a goat antibody, it can be detected usingan anti-mouse or anti-goat secondary antibody, respectively).

For the antibody regions discussed below (e.g., CDRs, framework regions,and the like), please refer to FIG. 10 (e.g., for sequence informationand SEQ ID NOs.).

In some cases, the first antibody of a subject two-way ELISA includes alight chain comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the aminoacid sequences set forth in SEQ ID NOs: 2-4, respectively, and a heavychain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively. In some cases,the first antibody includes the antigen binding region (e.g., the CDRs,and in some cases the framework region as well) of the C578 antibody. Insome cases, the first antibody is the C578 antibody. In some cases, thefirst antibody is a humanized version of the C578 antibody (e.g., theantibody includes the CDRs of the C578 antibody but is humanized).

In some cases, the first antibody includes a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 18-20, respectively, and a heavy chain comprising aCDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forthin SEQ ID NOs: 26-28, respectively. In some cases, the first antibodyincludes the antigen binding region (e.g., the CDRs, and in some casesthe framework region as well) of the 2A16 antibody. In some cases, thefirst antibody is the 2A16 antibody. In some cases, the first antibodyis a humanized version of the 2A16 antibody (e.g., the antibody includesthe CDRs of the 2A16 antibody but is humanized).

In some cases, the second antibody includes a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 2-4, respectively, and a heavy chain comprising a CDR-H1,CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQID NOs: 10-12, respectively. In some cases, the second antibody includesthe antigen binding region (e.g., the CDRs, and in some cases theframework region as well) of the C578 antibody. In some cases, thesecond antibody is the C578 antibody. In some cases, the second antibodyis a humanized version of the C578 antibody (e.g., the antibody includesthe CDRs of the C578 antibody but is humanized).

In some cases, the second antibody includes a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 18-20, respectively, and a heavy chain comprising aCDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forthin SEQ ID NOs: 26-28, respectively. In some cases, the second antibodyincludes the antigen binding region (e.g., the CDRs, and in some casesthe framework region as well) of the 2A16 antibody. In some cases, thesecond antibody is the 2A16 antibody. In some cases, the second antibodyis a humanized version of the 2A16 antibody (e.g., the antibody includesthe CDRs of the 2A16 antibody but is humanized).

In some cases, the first antibody includes a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 2-4, respectively, and a heavy chain comprising a CDR-H1,CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQID NOs: 10-12, respectively; and the second antibody includes a lightchain comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 18-20, respectively, and a heavychain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 26-28, respectively. In some cases,the first antibody includes a light chain comprising a CDR-L1, CDR-L2,and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs:18-20, respectively, and a heavy chain comprising a CDR-H1, CDR-H2, andCDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs:26-28, respectively; and the second antibody includes a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 2-4, respectively, and a heavy chaincomprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively. In some cases,the first antibody includes the antigen binding region (e.g., the CDRs,and in some cases the framework region as well) of the C578 antibody;and the second antibody includes the antigen binding region (e.g., theCDRs, and in some cases the framework region as well) of the 2A16antibody; or visa versa. In some cases, the first antibody is the C578antibody; and the second antibody is the 2A16 antibody; or visa versa.In some cases, the first antibody is a humanized version of the C578antibody (e.g., the antibody includes the CDRs of the C578 antibody butis humanized), and the second antibody is a humanized version of the2A16 antibody (e.g., the antibody includes the CDRs of the 2A16 antibodybut is humanized); or visa versa.

As such, in some cases, one of the first and second antibodies includesa light chain comprising a CDR-L1, CDR-L2, and CDR-L3 comprising theamino acid sequences set forth in SEQ ID NOs: 2-4, respectively, and aheavy chain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the aminoacid sequences set forth in SEQ ID NOs: 10-12, respectively; and theother of the first and second antibodies includes a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 18-20, respectively, and a heavychain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 26-28, respectively. In some cases,one of the first and second antibodies includes the antigen bindingregion (e.g., the CDRs, and in some cases the framework region as well)of the C578 antibody; and the other of the first and second antibodiesincludes the antigen binding region (e.g., the CDRs, and in some casesthe framework region as well) of the 2A16 antibody. In some cases, oneof the first and second antibodies is the C578 antibody; and the otherof the first and second antibodies is the 2A16 antibody. In some cases,one of the first and second antibodies is a humanized version of theC578 antibody (e.g., the antibody includes the CDRs of the C578 antibodybut is humanized), and the other of the first and second antibodies is ahumanized version of the 2A16 antibody (e.g., the antibody includes theCDRs of the 2A16 antibody but is humanized).

In some cases, a subject kit is a kit for measuring NMU in a sample(e.g., in a blood sample, in a serum sample, in a plasma sample, and thelike). In some cases, a subject kit includes a first anti-NMU antibodyand a second anti-NMU antibody, where one of the first and secondanti-NMU antibodies includes (i) a light chain comprising a CDR-L1,CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQID NOs: 2-4, respectively, and a heavy chain comprising a CDR-H1,CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQID NOs: 10-12, respectively; or (ii) a light chain comprising a CDR-L1,CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQID NOs: 18-20, respectively, and a heavy chain comprising a CDR-H1,CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQID NOs: 26-28, respectively.

In some cases, a subject kit includes a first anti-NMU antibody and asecond anti-NMU antibody, where one of the first and second anti-NMUantibodies includes a light chain comprising a CDR-L1, CDR-L2, andCDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 2-4,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 10-12,respectively; and the other of the first and second anti-NMU antibodiesincludes a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 18-20,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 26-28,respectively.

In some cases, a subject kit includes a first anti-NMU antibody and asecond anti-NMU antibody, where one of the first and second anti-NMUantibodies includes (i) the antigen binding region (e.g., the CDRs, andin some cases the framework region as well) of the C578 antibody; or(ii) the antigen binding region (e.g., the CDRs, and in some cases theframework region as well) of the 2A16 antibody. In some cases, a subjectkit includes a first anti-NMU antibody and a second anti-NMU antibody,where one of the first and second anti-NMU antibodies includes theantigen binding region (e.g., the CDRs, and in some cases the frameworkregion as well) of the C578 antibody; and the other of the first andsecond anti-NMU antibodies includes the antigen binding region (e.g.,the CDRs, and in some cases the framework region as well) of the 2A16antibody.

In some cases, a subject kit includes a first anti-NMU antibody and asecond anti-NMU antibody, where one of the first and second anti-NMUantibodies is (i) the C578 antibody; or (ii) the 2A16 antibody. In somecases, a subject kit includes a first anti-NMU antibody and a secondanti-NMU antibody, where one of the first and second anti-NMU antibodiesis the C578 antibody; and the other of the first and second anti-NMUantibodies is the 2A16 antibody.

In some cases, one or both of the first and second anti-NMU antibodiesare humanized (e.g., a humanized version of the C578 antibody and/or ahumanized version of the 2A16 antibody). In some cases, the firstanti-NMU antibody is immobilized on a solid surface (e.g., on thesurface of a well of a multi-well plate, on the surface of a bead,etc.). In some cases, the second anti-NMU antibody is labeled (e.g.,indirectly labeled and/or directly labeled, e.g., conjugated to anindirectly detectable label such as an enzyme, conjugated to a directlydetectable label such as a fluorophore or a fluorescent protein, and thelike).

In some cases, a subject two-way ELISA assay can detect (e.g., is usedto detect) NMU in a sample, where the NMU is present at a concentrationin a range of from 0.02 to 50 ng/mL (e.g., 0.02 to 40 ng/ml, 0.02 to 30ng/ml, 0.02 to 25 ng/ml, 0.02 to 20 ng/ml, 0.02 to 15 ng/ml, 0.05 to 50ng/ml, 0.05 to 40 ng/ml, 0.05 to 30 ng/ml, 0.05 to 25 ng/ml, 0.05 to 20ng/ml, 0.05 to 15 ng/ml, 0.1 to 50 ng/ml, 0.1 to 40 ng/ml, 0.1 to 30ng/ml, 0.1 to 25 ng/ml, 0.1 to 20 ng/ml, 0.1 to 15 ng/ml, 0.2 to 50ng/ml, 0.2 to 40 ng/ml, 0.2 to 30 ng/ml, 0.2 to 25 ng/ml, 0.2 to 20ng/ml, or 0.2 to 15 ng/ml). In some cases, a subject two-way ELISA assaycan detect (e.g., is used to detect) NMU in a sample, where the NMU ispresent at a concentration in a range of from 0.1 to 20 ng/ml.

Methods

Prediction Methods

Provided are prediction methods (e.g. predicting whether an individualwill develop diabetes, predicting whether an individual will developPDAC or pancreatitis, predicting whether an individual is in need oftherapy using an anti-NMU/NMUR agent, i.e., identifying an individualwho would benefit from administration of an anti-NMU/NMUR agent).Provided are diagnostic methods (e.g. predicting whether an individualhas diabetes, predicting/determining whether an individual has PDAC orpancreatitis, predicting whether an individual has type 3c diabetesmellitus (T3cDM), predicting whether an individual is in need of therapyusing an anti-NMU/NMUR agent, i.e., identifying an individual who wouldbenefit from administration of an anti-NMU/NMUR agent).

In some embodiments, a subject method is a method of predicting (e.g.,predicting that an individual will develop diabetes), and the methodincludes measuring NMU (e.g., using a two-way ELISA as described above).As such, in some embodiments, a subject method includes detecting (e.g.,measuring an amount of) NMU in sample (e.g., a biological sample such asa blood sample, serum sample, tumor sample, etc.). In some cases, NMU ismeasured via two-way ELISA (e.g., as described above). In some cases, ameasuring step (to measure NMU) is performed after a provocation stepsuch as an overnight fast, a glucose challenge (e.g., an oral bolus ofglucose), etc

Because the prediction and diagnostic methods are based on a measuredexpression level of NMU (protein and/or RNA) in a sample, the predictionmethods include a step of measuring, e.g., measuring an expression levelof an NMU expression product in a biological sample from an individual(e.g., measuring a protein expression level, i.e., the amount of NMU ina sample; measuring an mRNA expression level, i.e., the amount of mRNAencoding NMU in a sample). Thus, the present disclosure providescompositions for measuring neuromedin U (NMU) in a sample (e.g., abiological sample such as a blood sample, a serum sample, a biopsy,etc.).

A biomarker is a molecular entity (e.g., an expression product such asmRNA, protein, etc.) whose representation in a sample correlates (eitherpositively or negatively) with a particular state. For example, anexpression level of NMU (protein) in the serum correlates with whetheran individual is likely to develop diabetes. As demonstrated in theexamples of the present disclosure, the inventors have identified NMU asa biomarker positively associated with increased likelihood that a givenindividual will develop diabetes (e.g., type 2 Diabetes (T2DM)). Forexample, an increased level of NMU in the serum is predictive that anindividual will develop diabetes. In some cases, the individual issuspected of having an increased risk of developing diabetes (e.g., theindividual has a family history of diabetes, is overweight, and/or isobese) prior to the measuring step. In some cases, a subject methodincludes (a) measuring an expression level of NMU (e.g., mRNA, protein)in a biological sample from an individual (e.g., a blood sample, abiopsy, a serum sample), (b) determining that the measured expressionlevel of NMU is greater than or equal to a reference value; and (c)predicting that the individual will develop diabetes. In some cases, themeasuring step is performed using an anti-NMU antibody (or fragmentthereof) disclosed herein. In some cases, the measuring step isperformed using a two-way ELISA disclosed herein.

In some cases, NMU can be used as a biomarker positively associated withincreased likelihood that a given individual will develop PDAC. In somecases, the individual is suspected of having an increased risk ofdeveloping PDAC (e.g., the individual may have a family history of PDAC,may have pancreatitis, may already have diabetes, etc.). As such in somecases, a subject method includes (a) measuring an expression level ofNMU (e.g., mRNA, protein) in a biological sample from an individual(e.g., a blood sample, a biopsy), (b) determining that the measuredexpression level of NMU is greater than or equal to a reference value;and (c) predicting that the individual will develop PDAC. In some cases,the measuring step is performed using an anti-NMU antibody (or fragmentthereof) disclosed herein. In some cases, the measuring step isperformed using a two-way ELISA disclosed herein.

In some cases, NMU can be used as a biomarker positively associated withincreased likelihood that a given individual will develop PDAC orpancreatitis (e.g., recurrent acute pancreatitis and/or chronicpancreatitis). In some cases, the individual is suspected of having anincreased risk of developing PDAC or pancreatitis (e.g., recurrent acutepancreatitis and/or chronic pancreatitis) (e.g., the individual may havea family history of PDAC or pancreatitis). As such in some cases, asubject method includes (a) measuring an expression level of NMU (e.g.,mRNA, protein) in a biological sample from an individual (e.g., a bloodsample, a biopsy), (b) determining that the measured expression level ofNMU is greater than or equal to a reference value; and (c) predictingthat the individual will develop PDAC or pancreatitis (e.g., recurrentacute pancreatitis and/or chronic pancreatitis). In some cases, themeasuring step is performed using an anti-NMU antibody (or fragmentthereof) disclosed herein. In some cases, the measuring step isperformed using a two-way ELISA disclosed herein.

In some cases, NMU can be used as a biomarker positively associated withincreased likelihood that a given individual will be responsive to(e.g., is in need of) administration of a subject anti-NMU/NMUR agent(e.g., an anti-NMU antibody). In some cases, the individual is suspectedof being an individual will be responsive to (e.g., is in need of)administration of a subject anti-NMU/NMUR agent (e.g., an anti-NMUantibody) (e.g., in some cases, the individual is overweight, obese, hasdiabetes, has type 2 diabetes, has type 3c diabetes, has PDAC, hasrecurring acute pancreatitis, has chronic pancreatitis, and/or hascancer cachexia. As such in some cases, a subject method includes (a)measuring an amount of NMU present in a blood sample from an individual(e.g., a serum sample), (b) determining that the amount of NMU presentin the blood sample is greater than or equal to a reference value (e.g.,see below); and (c) predicting that the individual would benefit fromadministration of an anti-NMU/NMUR agent. In some cases, the measuringstep is performed using an anti-NMU antibody (or fragment thereof)disclosed herein. In some cases, the measuring step is performed using atwo-way ELISA disclosed herein.

The terms “assaying” and “measuring” are used herein to include thephysical steps of manipulating a biological sample (e.g., blood sample,serum sample, cell sample, biopsy, and the like) to generate datarelated to a sample (e.g., measuring an expression level in a biologicalsample). In practicing the subject methods, the expression level of aNMU expression product (e.g., mRNA, protein) can be measured (e.g., theexpression level in a cell, in a population of cells, in a biologicalsample from an individual, and the like). The expression level(s) can bemeasured by any convenient method. For example, an RNA expression levelcan be measured by measuring the levels/amounts of one or more nucleicacid transcripts, e.g. mRNAs, of NMU. Protein expression levels of NMUcan be detected by measuring the levels/amounts of the NMU protein(e.g., using a two-way ELISA disclosed herein). In some cases, measuringis performed using an anti-NMU antibody (or fragment thereof) disclosedherein. In some cases, the measuring step is performed using a two-wayELISA disclosed herein.

“Measuring” can be used to determine whether the measured expressionlevel is less than, greater than, “less than or equal to”, or “greaterthan or equal to” a particular threshold, (the threshold can bepre-determined or can be determined by assaying a control sample), suchas a reference value. Measuring can mean determining a quantitativevalue (using any convenient metric) that represents the level ofexpression (i.e., expression level, e.g., the amount of protein and/orRNA, e.g., mRNA) of a particular expression product (e.g., a NMUexpression product). The level of expression can be expressed inarbitrary units associated with a particular assay (e.g., fluorescenceunits, e.g., mean fluorescence intensity (MFI), threshold cycle (C_(t)),quantification cycle (C_(q)), and the like), or can be expressed as anabsolute value with defined units (e.g., number of mRNA transcripts,number of protein molecules, concentration of protein, etc.).

An expression level (i.e., level of expression) can be a raw measuredvalue, or can be a normalized and/or weighted value derived from the rawmeasured value. The terms “expression level” and “measured expressionlevel” are used herein to encompass raw measured values as well asvalues that have manipulated in some way (e.g., normalized and/orweighted). In some cases, a normalized expression level is a measuredexpression level of an expression product from a sample where the rawmeasured value for the expression product has been normalized. Forexample, the expression level of an expression product (e.g., an RNAencoding NMU, a NMU protein) can be compared to the expression level ofone or more other expression products (e.g., the expression level of ahousekeeping gene/protein, the averaged expression levels of multiplegenes/proteins, etc.) to derive a normalized value that represents anormalized expression level. Methods of normalization will be known toone of ordinary skill in the art and any convenient normalization methodcan be used. The specific metric (or units) chosen is not crucial aslong as the same units are used (or conversion to the same units isperformed) when evaluating multiple markers and/or multiple biologicalsamples (e.g., samples from multiple individuals or multiple samplesfrom the same individual).

Measuring Protein

An expression level of an expression product (e.g., an expressionproduct of NMU) may be measured by detecting (e.g., in a cell extract,in a fixed cell, in living cell, in a biological sample, in a bloodsample, in a serum sample, etc.) the amount or level of one or moreproteins (e.g., NMU) or a fragment thereof. For measuring a proteinlevel, the amount or level of protein the sample (e.g., in a cellextract, in a fixed cell, in living cell, in a biological sample, in ablood sample, in a serum sample, etc.) is determined. In some instances,the concentration of one or more additional proteins may also bemeasured, and the measured expression level compared to the level of theone or more additional proteins to provide a normalized value for themeasured expression level. In some embodiments, the measured expressionlevel is a relative value calculated by comparing the level of oneprotein relative to another protein. In other embodiments theconcentration is an absolute measurement (e.g., weight/volume orweight/weight).

The expression level of a protein (e.g., NMU) may be measured bydetecting in a sample the amount or level of one or moreproteins/polypeptides or fragments thereof. The terms “polypeptide,”“peptide” and “protein” are used interchangeably herein to refer to apolymer of amino acid residues. “Polypeptide” refers to a polymer ofamino acids (amino acid sequence) and does not refer to a specificlength of the molecule. Thus peptides and oligopeptides are includedwithin the definition of polypeptide. In some cases, cells and/orexosomes are removed from a biological sample (e.g., via centrifugation,via adhering cells to a dish or to plastic, etc.) prior to measuring theexpression level (e.g., measuring in a serum sample from which exosomeshave been removed).

When protein levels are to be detected, any convenient protocol formeasuring protein levels may be employed. Examples of methods forassaying protein levels include but are not limited to enzyme-linkedimmunosorbent assay (ELISA) (e.g., a two-way ELISA as disclosed herein),mass spectrometry, proteomic arrays, xMAP™ microsphere technology, flowcytometry, western blotting, immunohistochemistry, and the like. In somecases, an anti-NMU antibody described herein is used for measuring alevel of NMU protein in a sample (e.g., a serum sample).

Some protein detection methods are antibody-based methods (e.g., asubject two-way ELISA). The term “antibody” is used in the broadestsense and specifically covers monoclonal antibodies (including fulllength monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired biological activity.

Measuring RNA

An expression level of an expression product (e.g., an expressionproduct of NMU) may be measured by detecting (e.g., in a cell extract,in a fixed cell, in a living cell, in a biological sample, in a biopsysample, etc.) the amount or level of one or more RNA transcripts or afragment thereof encoded by the gene of interest (NMU). For measuringRNA levels, the amount or level of an RNA in the sample is determined,e.g., the expression level of an mRNA. In some instances, the expressionlevel of one or more additional RNAs may also be measured, and the levelof biomarker expression compared to the level of the one or moreadditional RNAs to provide a normalized value for the biomarkerexpression level.

The expression level of nucleic acids in the sample may be detectedusing any convenient protocol. A number of exemplary methods formeasuring RNA (e.g., mRNA) expression levels (e.g., expression level ofa nucleic acid biomarker) in a sample are known by one of ordinary skillin the art, such as those methods employed in the field of differentialgene expression analysis, and any convenient method can be used.Exemplary methods include, but are not limited to: hybridization-basedmethods (e.g., Northern blotting, array hybridization (e.g.,microarray); in situ hybridization; in situ hybridization followed byFACS; and the like)(Parker & Barnes, Methods in Molecular Biology106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques13:852-854 (1992)); PCR-based methods (e.g., reverse transcription PCR(RT-PCR), quantitative RT-PCR (qRT-PCR), real-time RT-PCR, etc.)(Weis etal., Trends in Genetics 8:263-264 (1992)); nucleic acid sequencingmethods (e.g., Sanger sequencing, Next Generation sequencing (i.e.,massive parallel high throughput sequencing, e.g., Illumina's reversibleterminator method, Roche's pyrosequencing method (454), LifeTechnologies' sequencing by ligation (the SOLID platform), LifeTechnologies' Ion Torrent platform, single molecule sequencing, etc.);nanopore based sequencing methods; and the like.

In some embodiments, the biological sample can be assayed directly. Insome embodiments, nucleic acid of the biological sample is amplified(e.g., by PCR) prior to assaying. As such, techniques such as PCR(Polymerase Chain Reaction), RT-PCR (reverse transcriptase PCR), qRT-PCR(quantitative RT-PCR, real time RT-PCR), etc. can be used prior to thehybridization methods and/or the sequencing methods discussed above.

As noted above, gene expression in a sample can be detected usinghybridization analysis, which is based on the specificity of nucleotideinteractions. Oligonucleotides or cDNA can be used to selectivelyidentify or capture DNA or RNA of specific sequence composition, and theamount of RNA or cDNA hybridized to a known capture sequence determinedqualitatively or quantitatively, to provide information about therelative representation of a particular message within the pool ofcellular messages in a sample. Hybridization analysis can be designed toallow for concurrent screening of the relative expression of hundreds tothousands of genes by using, for example, array-based technologieshaving high density formats, including filters, microscope slides, ormicrochips, or solution-based technologies that use spectroscopicanalysis.

Hybridization to arrays may be performed, where the arrays can beproduced according to any suitable methods known in the art. Forexample, methods of producing large arrays of oligonucleotides aredescribed in U.S. Pat. Nos. 5,134,854, and 5,445,934 usinglight-directed synthesis techniques. Using a computer controlled system,a heterogeneous array of monomers is converted, through simultaneouscoupling at a number of reaction sites, into a heterogeneous array ofpolymers. Alternatively, microarrays are generated by deposition ofpre-synthesized oligonucleotides onto a solid substrate, for example asdescribed in PCT published application no. WO 95/35505.

Methods for collection of data from hybridization of samples with anarray are also well known in the art. For example, the polynucleotidesof the cell samples can be generated using a detectable fluorescentlabel, and hybridization of the polynucleotides in the samples detectedby scanning the microarrays for the presence of the detectable label.Methods and devices for detecting fluorescently marked targets ondevices are known in the art. Generally, such detection devices includea microscope and light source for directing light at a substrate. Aphoton counter detects fluorescence from the substrate, while an x-ytranslation stage varies the location of the substrate. A confocaldetection device that can be used in the subject methods is described inU.S. Pat. No. 5,631,734. A scanning laser microscope is described inShalon et al., Genome Res. (1996) 6:639. A scan, using the appropriateexcitation line, is performed for each fluorophore used. The digitalimages generated from the scan are then combined for subsequentanalysis. For any particular array element, the ratio of the fluorescentsignal from one sample is compared to the fluorescent signal fromanother sample, and the relative signal intensity determined.

Methods for analyzing the data collected from hybridization to arraysare well known in the art. For example, where detection of hybridizationinvolves a fluorescent label, data analysis can include the steps ofdetermining fluorescent intensity as a function of substrate positionfrom the data collected, removing outliers, i.e. data deviating from apredetermined statistical distribution, and calculating the relativebinding affinity of the targets from the remaining data. The resultingdata can be displayed as an image with the intensity in each regionvarying according to the binding affinity between targets and probes.

One representative and convenient type of protocol for measuring mRNAlevels is array-based gene expression profiling. Such protocols arehybridization assays in which a nucleic acid that displays “probe”nucleic acids for each of the genes to be assayed/profiled in theprofile to be generated is employed. In these assays, a sample of targetnucleic acids is first prepared from the initial nucleic acid samplebeing assayed, where preparation may include labeling of the targetnucleic acids with a label, e.g., a member of signal producing system.Following target nucleic acid sample preparation, the sample iscontacted with the array under hybridization conditions, wherebycomplexes are formed between target nucleic acids that are complementaryto probe sequences attached to the array surface. The presence ofhybridized complexes is then detected, either qualitatively orquantitatively.

Specific hybridization technology which may be practiced to generate theexpression profiles employed in the subject methods includes thetechnology described in U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633;5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464;5,547,839; 5,580,732; 5,661,028; 5,800,992; the disclosures of which areherein incorporated by reference; as well as WO 95/21265; WO 96/31622;WO 97/10365; WO 97/27317; EP 373 203; and EP 785 280. In these methods,an array of “probe” nucleic acids that includes a probe for each of thephenotype determinative genes whose expression is being assayed iscontacted with target nucleic acids as described above. Contact iscarried out under hybridization conditions, e.g., stringenthybridization conditions, and unbound nucleic acid is then removed. Theterm “stringent assay conditions” as used herein refers to conditionsthat are compatible to produce binding pairs of nucleic acids, e.g.,surface bound and solution phase nucleic acids, of sufficientcomplementarity to provide for the desired level of specificity in theassay while being less compatible to the formation of binding pairsbetween binding members of insufficient complementarity to provide forthe desired specificity. Stringent assay conditions are the summation orcombination (totality) of both hybridization and wash conditions.

The resultant pattern of hybridized nucleic acid provides informationregarding expression for each of the genes that have been probed, wherethe expression information is in terms of whether or not the gene isexpressed and, typically, at what level, where the expression data,i.e., expression profile (e.g., in the form of a transcriptosome), maybe both qualitative and quantitative. Pattern analysis can be performedmanually, or can be performed using a computer program. Methods forpreparation of substrate matrices (e.g., arrays), design ofoligonucleotides for use with such matrices, labeling of probes,hybridization conditions, scanning of hybridized matrices, and analysisof patterns generated, including comparison analysis, are described in,for example, U.S. Pat. No. 5,800,992.

Alternatively, non-array based methods for quantitating the level of oneor more nucleic acids in a sample may be employed. These include thosebased on amplification protocols, e.g., Polymerase Chain Reaction(PCR)-based assays, including quantitative PCR, reverse-transcriptionPCR (RT-PCR), real-time PCR, quantitative RT-PCR (qRT-PCR), and thelike, e.g. TaqMan® RT-PCR, SYBR green; MassARRAY® System, BeadArray®technology, and Luminex technology; and those that rely uponhybridization of probes to filters, e.g. Northern blotting and in situhybridization. Other non-amplified methods of analysis include digitalbar-coding, e.g. NanoString nCounter Analysis System which is a digitalcolor-coded barcode technology based on direct multiplexed measurementof gene expression. The technology uses molecular “barcodes” and singlemolecule imaging to detect and count hundreds of unique transcripts in asingle reaction. Each color-coded barcode is attached to a singletarget-specific probe corresponding to a gene of interest. Mixedtogether with controls, they form a multiplexed CodeSet.

Examples of some of the nucleic acid sequencing methods listed above aredescribed in the following references: Margulies et al (Nature 2005 437:376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9);Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform. 200910:609-18); Fox et al (Methods Mol Biol. 2009; 553:79-108); Appleby etal (Methods Mol Biol. 2009; 513:19-39); Soni et al Clin Chem 53:1996-2001 2007; and Morozova (Genomics. 2008 92:255-64), which areincorporated by reference for the general descriptions of the methodsand the particular steps of the methods, including starting products,reagents, and final products for each of the steps.

For measuring mRNA levels, the starting material can be RNA or poly A+RNA (e.g., isolated from a biological sample, from a suspension ofcells, etc.). General methods for mRNA extraction are known in the artand are disclosed in standard textbooks of molecular biology, includingAusubel et al., Current Protocols of Molecular Biology, John Wiley andSons (1997). RNA isolation (e.g., mRNA isolation) can be performed usingany convenient protocol. For example, RNA isolation can be performedusing a purification kit, buffer set and protease from commercialmanufacturers, according to the manufacturer's instructions. Forexample, RNA from cell suspensions can be isolated using Qiagen RNeasymini-columns, and RNA from cell suspensions or homogenized tissuesamples can be isolated using the TRIzol reagent-based kits(Invitrogen), MasterPure™ Complete DNA and RNA Purification Kit(EPICENTRE™, Madison, Wis.), Paraffin Block RNA Isolation Kit (Ambion,Inc.) or RNA Stat-60 kit (Tel-Test).

Reference Value

A measured NMU expression level (e.g., NMU protein, NMU encoding mRNA)can be determined in a number of different ways. For example, in somecases, a NMU expression level is measured in a sample (e.g., theconcentration of NMU in sample such as serum sample is measured) and iscompared to a reference value.

In some cases, the expression level (e.g., the number of transcripts,the concentration of NMU protein in a sample, and the like) of an NMUexpression product (e.g., RNA, protein) in a biological sample from anindividual who is predicted to develop diabetes, PDAC, and/orpancreatitis is greater than a reference value (e.g., an expressionlevel of an NMU expression product in one or more biological samplesfrom one or more control individuals who do not have diabetes, PDAC,and/or pancreatitis; a value, e.g., an average, derived from theexpression level of an NMU expression product in a biological samplefrom multiple control individuals who do not have diabetes, PDAC, and/orpancreatitis; etc.).

For example, the expression level (e.g., the concentration of protein,the number of transcripts, the concentration of NMU protein in a sample,and the like) of an NMU expression product (e.g., RNA, protein) in abiological sample from an individual who is predicted to developdiabetes, PDAC, and/or pancreatitis can be 1.1-fold or more (e.g.,1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more,2-fold or more, 2.5-fold or more, 3-fold or more, 4-fold or more, 5-foldor more, 7.5-fold or more, or 10-fold or more) greater than a referencevalue (e.g., an expression level of an NMU expression product in one ormore biological samples from one or more control individuals who do nothave diabetes, PDAC, and/or pancreatitis; a value, e.g., an average,derived from the expression level of an NMU expression product in abiological sample from multiple control individuals who do not havediabetes, PDAC, and/or pancreatitis; etc.).

In some cases, the expression level (e.g., the number of transcripts,the concentration of NMU protein in a sample, and the like) of an NMUexpression product (e.g., RNA, protein) in a biological sample from anindividual who would benefit from administration of a subjectanti-NMU/NMUR agent (e.g., an anti-NMU antibody) is greater than areference value (e.g., an expression level of an NMU expression productin one or more biological samples from one or more control individualswho are not in need of (or who would not benefit from) administration ofa subject anti-NMU/NMUR agent (e.g., an anti-NMU antibody), e.g., anaverage, derived from the expression level of an NMU expression productin a biological sample from multiple control individuals who are not inneed of (or who would not benefit from) administration of a subjectanti-NMU/NMUR agent (e.g., an anti-NMU antibody)).

For example, the expression level (e.g., the concentration of protein,the number of transcripts, the concentration of NMU protein in a sample,and the like) of an NMU expression product (e.g., RNA, protein) in abiological sample from an individual who would benefit fromadministration of a subject anti-NMU/NMUR agent (e.g., an anti-NMUantibody) can be 1.1-fold or more (e.g., 1.2-fold or more, 1.3-fold ormore, 1.4-fold or more, 1.5-fold or more, 2-fold or more, 2.5-fold ormore, 3-fold or more, 4-fold or more, 5-fold or more, 7.5-fold or more,or 10-fold or more) greater than a reference value (e.g., an expressionlevel of an NMU expression product in one or more biological samplesfrom one or more control individuals who are not in need of (or whowould not benefit from) administration of a subject anti-NMU/NMUR agent(e.g., an anti-NMU antibody), e.g., an average, derived from theexpression level of an NMU expression product in a biological samplefrom multiple control individuals who are not in need of (or who wouldnot benefit from) administration of a subject anti-NMU/NMUR agent (e.g.,an anti-NMU antibody)).

In some cases, the predicting and/or diagnosing is based on a measuredexpression level of NMU (e.g., protein, mRNA) in combination with one ormore clinical measurements (e.g., a meal tolerance test (MTT), patientweight, an insulin measurement, a test of insulin sensitivity, a measureof glucose level, etc.).

Generating a Report

In some cases, a subject method (e.g., any of the screening methodsdescribed above) includes a step of generating a report (e.g., a reportthat the test agent is a candidate agent for treating obesity and/ordiabetes).

A “report,” as described herein, is an electronic or tangible documentwhich includes report elements that provide information of interestrelating to the results and/or assessments of such results of a subjectmethod (e.g., a prediction and/or diagnostic method). In someembodiments, a subject report includes a measured expression level asdiscussed in greater detail above (e.g., a raw value, a normalizedvalue, a normalized and weighted value, etc.) (e.g., an expression levelof a NMU expression product such as a NMU protein expression leveland/or a NMU-encoding mRNA expression level). In some embodiments, asubject report includes a NMU expression level. In some cases, a subjectreport includes an assessment (e.g. a determination of whether an NMUexpression product such as an NMU protein or NMU encoding mRNA iselevated in a sample, e.g., relative to a reference value).

A subject report can be completely or partially electronicallygenerated. A subject report can include one or more of: 1) the assayused to measure the NMU expression level (e.g., a subject two-wayELISA); 2) raw data; 3) details of how an expression level wascalculated from raw data; 4) a value associated with whether (and if sohow much) a NMU expression level is elevated relative to a reference; 5)information about the biological sample tested; 6) information about theindividual from whom the biological sample was collected; and the like.Thus, the subject methods may include a step of generating or outputtinga report, which report can be provided in the form of an electronicmedium (e.g., an electronic display on a computer monitor), or in theform of a tangible medium (e.g., a report printed on paper or othertangible medium). Any form of report may be provided.

It will also be readily appreciated that the reports can includeadditional elements or modified elements. For example, where electronic,the report can contain hyperlinks which point to internal or externaldatabases which provide more detailed information about selectedelements of the report. When in electronic format, the report isrecorded on a suitable physical medium, such as a computer readablemedium, e.g., in a computer memory, zip drive, CD, DVD, etc.

Treatment Methods

In some embodiments, a subject method is a treatment method (e.g., usingan anti-NMU/NMUR agent, e.g., as described above). For example in somecases, a subject method is a method of treating an individual in needthereof (e.g., an individual with elevated NMU levels, e.g., serum NMUlevels). In some cases, a subject method is a method of increasingcirculating insulin in an individual. In some cases, the individual hasdiabetes, or is suspected of having an increased risk of developingdiabetes (e.g., the individual is obese and/or has a family history thatincludes diabetics). In some cases, the individual has a diseaseselected from: cystic fibrosis, familial pancreatitis, idiopathicpancreatitis, chronic pancreatitis, PDAC, type 3c diabetes mellitus,type 2 diabetes, late stage pancreatic cancer, and cancer cachexia.

Treatment methods provided herein include a step of administering ananti-NMU/NMUR agent (e.g., an anti-NMU antibody, an RNAi agent thattargets NMUR1) to an individual. Details related to suitableanti-NMU/NMUR agents can be found above.

For example, any of the above discussed anti-NMU antibodies can be usedas an anti-NMU/NMUR agent. Thus, in some cases, a subject anti-NMUantibody (or antigen binding fragment thereof) includes a light chaincomprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acidsequences set forth in SEQ ID NOs: 18-20, respectively. In some cases, asubject anti-NMU antibody (or antigen binding fragment thereof) includesa heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising theamino acid sequences set forth in SEQ ID NOs: 26-28, respectively. Insome cases, a subject anti-NMU antibody (or antigen binding fragmentthereof) includes a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 18-20,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 26-28,respectively. In some cases, a subject anti-NMU antibody (or antigenbinding fragment thereof) includes the antigen binding region (e.g., theCDRs, and in some cases the framework region as well) of the 2A16antibody. In some cases, a subject anti-NMU antibody is the 2A16antibody. In some cases, a subject anti-NMU antibody is a humanizedversion of the 2A16 antibody (e.g., the antibody includes the CDRs ofthe 2A16 antibody but is humanized).

As such, in some cases, a subject humanized anti-NMU antibody (orantigen binding fragment thereof) includes a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 18-20, respectively. In some cases, a subject humanizedanti-NMU antibody (or antigen binding fragment thereof) includes a heavychain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 26-28, respectively. In some cases, asubject humanized anti-NMU antibody (or antigen binding fragmentthereof) includes a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 18-20,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 26-28,respectively. In some cases, a subject humanized anti-NMU antibody (orantigen binding fragment thereof) includes the antigen binding region(e.g., the CDRs, and in some cases the framework region as well) of the2A16 antibody. In some cases, a subject humanized anti-NMU antibody isthe 2A16 antibody. In some cases, a subject humanized anti-NMU antibodyis a humanized version of the 2A16 antibody (e.g., the antibody includesthe CDRs of the 2A16 antibody but is humanized).

In some cases, a subject anti-NMU antibody (or antigen binding fragmentthereof) includes a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 2-4,respectively. In some cases, a subject anti-NMU antibody (or antigenbinding fragment thereof) includes a heavy chain comprising a CDR-H1,CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQID NOs: 10-12, respectively. In some cases, a subject anti-NMU antibody(or antigen binding fragment thereof) includes a light chain comprisinga CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences setforth in SEQ ID NOs: 2-4, respectively, and a heavy chain comprising aCDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forthin SEQ ID NOs: 10-12, respectively. In some cases, a subject anti-NMUantibody (or antigen binding fragment thereof) includes the antigenbinding region (e.g., the CDRs, and in some cases the framework regionas well) of the C578 antibody. In some cases, a subject anti-NMUantibody is the C578 antibody. In some cases, a subject anti-NMUantibody is a humanized version of the C578 antibody (e.g., the antibodyincludes the CDRs of the C578 antibody but is humanized).

As such, in some cases, a subject humanized anti-NMU antibody (orantigen binding fragment thereof) includes a light chain comprising aCDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forthin SEQ ID NOs: 2-4, respectively. In some cases, a subject humanizedanti-NMU antibody (or antigen binding fragment thereof) includes a heavychain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences set forth in SEQ ID NOs: 10-12, respectively. In some cases, asubject humanized anti-NMU antibody (or antigen binding fragmentthereof) includes a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 2-4,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 10-12,respectively. In some cases, a subject humanized anti-NMU antibody (orantigen binding fragment thereof) includes the antigen binding region(e.g., the CDRs, and in some cases the framework region as well) of theC578 antibody. In some cases, a subject humanized anti-NMU antibody isthe C578 antibody. In some cases, a subject humanized anti-NMU antibodyis a humanized version of the C578 antibody (e.g., the antibody includesthe CDRs of the C578 antibody but is humanized).

Formulations

An anti-NMU/NMUR agent (e.g., an anti-NMU antibody) can be prepared as adosage unit, with a pharmaceutically acceptable excipient, withpharmaceutically acceptable salts and esters, etc. Compositions can beprovided as pharmaceutical compositions.

Pharmaceutical Compositions.

Suitable anti-NMU/NMUR agents (e.g., one or more anti-NMU antibodies)can be provided in pharmaceutical compositions suitable for therapeuticuse, e.g. for human treatment. In some embodiments, pharmaceuticalcompositions of the present disclosure include one or more therapeuticentities of the present disclosure (e.g., one or more anti-NMUantibodies) and can include a pharmaceutically acceptable carrier, apharmaceutically acceptable salt, a pharmaceutically acceptableexcipient, and/or esters or solvates thereof. In some embodiments, theuse of an anti-NMU/NMUR agent (e.g., anti-NMU antibody) includes use incombination with another therapeutic agent (e.g., another agent forpreventing or treating diabetes, preventing, controlling, or treatingobesity, preventing or treating a cancer such as PDAC, and the like).Therapeutic formulations comprising an anti-NMU/NMUR agent (e.g., ananti-NMU antibody) can be prepared by mixing the agent(s) having thedesired degree of purity with a physiologically acceptable carrier, apharmaceutically acceptable salt, an excipient, and/or a stabilizer(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980))(e.g., in the form of lyophilized formulations or aqueous solutions). Acomposition having an anti-NMU/NMUR agent (e.g., an anti-NMU antibody)can be formulated, dosed, and administered in a fashion consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom(s) thereofand/or may be therapeutic in terms of a partial or completestabilization or cure for a disease and/or adverse effect attributableto the disease. The term “treatment” encompasses any treatment of adisease in a mammal, particularly a human, and includes: (a) preventingthe disease and/or symptom(s) from occurring in a subject who may bepredisposed to the disease or symptom but has not yet been diagnosed ashaving it; (b) inhibiting the disease and/or symptom(s), i.e., arrestingtheir development; or (c) relieving the disease symptom(s), i.e.,causing regression of the disease and/or symptom(s). Those in need oftreatment include those already inflicted (e.g., those with obesity,diabetes, pancreatic cancer, PDAC, etc.) as well as those in whichprevention is desired (e.g., those with increased susceptibility todiabetes and/or cancer such as PDAC, etc.).

A therapeutic treatment is one in which the subject is inflicted (e.g.,has the disease) prior to administration and a prophylactic treatment isone in which the subject is not yet inflicted (does not yet have thedisease) prior to administration. In some embodiments, the subject hasan increased likelihood of becoming inflicted or is suspected of beinginflicted prior to treatment. In some embodiments, the subject issuspected of having an increased likelihood of becoming inflicted. Forexample, in some cases, the individual is obese, has a family history ofobesity and/or diabetes, has a family history of pancreatic cancer suchas PDAC, etc.

A “therapeutically effective dose” or “therapeutic dose” is an amountsufficient to effect desired clinical results (i.e., achieve therapeuticefficacy), e.g., increased insulin output. A therapeutically effectivedose can be administered in one or more administrations. For purposes ofthis disclosure, a therapeutically effective dose of an anti-NMU/NMURagent (e.g., an anti-NMU antibody) is an amount that is sufficient topalliate, ameliorate, stabilize, reverse, prevent, slow or delay theprogression of the disease state (e.g., diabetes, obesity, PDAC). Thus,in some cases, a therapeutically effective dose of an anti-NMU/NMURagent (e.g., an anti-NMU antibody) reduces the binding of circulatingNMU to its receptor on the surface of cells at an effective dose forincreasing insulin output in an individual.

A single therapeutically effective dose or a series of therapeuticallyeffective doses would be able to achieve a desired result in anindividual (e.g., increase circulating level of insulin). Atherapeutically effective dose of an anti-NMU/NMUR agent (e.g., ananti-NMU antibody) can depend on the specific agent used, and in somecases can be 0.5 mg/kg body weight or more (e.g., 1 mg/kg or more, 2mg/kg or more, 3 mg/kg or more, 4 mg/kg or more, 5 mg/kg or more, 6mg/kg or more, 7 mg/kg or more, 8 mg/kg or more, 9 mg/kg or more, 10mg/kg or more, 15 mg/kg or more, 20 mg/kg or more, 25 mg/kg or more, 30mg/kg or more, 35 mg/kg or more, or 40 mg/kg or more) for each agent.

In some cases, a therapeutically effective dose of an anti-NMU/NMURagent (e.g., an anti-NMU antibody) can be in a range of from 0.5 mg/kgto 100 mg/kg (e.g., from 0.5 to 90 mg/kg, from 0.5 to 90 mg/kg, from 0.5to 80 mg/kg, from 0.5 to 70 mg/kg, from 0.5 to 60 mg/kg, from 0.5 to 50mg/kg, from 0.5 to 40 mg/kg, from 0.5 to 30 mg/kg, from 0.5 to 20 mg/kg,from 0.5 to 10 mg/kg, from 1 to 100 mg/kg, from 1 to 90 mg/kg, from 1 to90 mg/kg, from 1 to 80 mg/kg, from 1 to 70 mg/kg, from 1 to 60 mg/kg,from 1 to 50 mg/kg, from 1 to 40 mg/kg, from 1 to 30 mg/kg, from 1 to 20mg/kg, from 1 to 10 mg/kg, from 3 to 100 mg/kg, from 3 to 90 mg/kg, from3 to 90 mg/kg, from 3 to 80 mg/kg, from 3 to 70 mg/kg, from 3 to 60mg/kg, from 3 to 50 mg/kg, from 3 to 40 mg/kg, from 3 to 30 mg/kg, from3 to 20 mg/kg, from 3 to 10 mg/kg, from 5 to 100 mg/kg, from 5 to 90mg/kg, from 5 to 90 mg/kg, from 5 to 80 mg/kg, from 5 to 70 mg/kg, from5 to 60 mg/kg, from 5 to 50 mg/kg, from 5 to 40 mg/kg, from 5 to 30mg/kg, from 5 to 20 mg/kg, from 5 to 10 mg/kg, from 10 to 100 mg/kg,from 10 to 90 mg/kg, from 10 to 90 mg/kg, from 10 to 80 mg/kg, from 10to 70 mg/kg, from 10 to 60 mg/kg, from 10 to 50 mg/kg, from 10 to 40mg/kg, from 10 to 30 mg/kg, from 10 to 20 mg/kg, from 20 to 100 mg/kg,from 20 to 90 mg/kg, from 20 to 90 mg/kg, from 20 to 80 mg/kg, from 20to 70 mg/kg, from 20 to 60 mg/kg, from 20 to 50 mg/kg, from 20 to 40mg/kg, or from 20 to 30 mg/kg) for each agent.

In some cases, a therapeutically effective dose of an anti-NMU/NMURagent (e.g., an anti-NMU antibody) can be in a range of from 5 mg/kg to50 mg/kg (e.g., from 5 to 40 mg/kg, from 5 to 30 mg/kg, from 5 to 20mg/kg, from 10 to 50 mg/kg, from 10 to 40 mg/kg, from 10 to 30 mg/kg, orfrom 10 to 20 mg/kg) for each agent. In some cases, a therapeuticallyeffective dose of an anti-NMU/NMUR agent (e.g., an anti-NMU antibody)can be in a range of from 10 mg/kg to 40 mg/kg (e.g., from 10 to 35mg/kg, or from 10 to 30 mg/kg) for each agent.

The dose required to achieve a desired result can be proportional to theamount of time between doses and inversely proportional to the number ofdoses administered. Thus, as the frequency of dosing increases, therequired dose decreases. The optimization of dosing strategies will bereadily understood and practiced by one of ordinary skill in the art.

Dosage and frequency may vary depending on the half-life of theanti-NMU/NMUR agent (e.g., an anti-NMU antibody) in the patient. It willbe understood by one of skill in the art that such guidelines will beadjusted for the molecular weight of the active agent, e.g. in the useof antibody fragments, in the use of antibody conjugates, in the use ofRNAi agents, etc. The dosage may also be varied for localizedadministration, e.g. intranasal, inhalation, etc., or for systemicadministration, e.g. i.m., i.p., i.v., and the like.

Co-Administration

Two of the above described anti-NMU antibodies can be co-administered,and antibody-based NMU inhibition could be readily combined withstandard therapies for diseases like obesity and diabetes. As such, insome cases, a subject anti-NMU/NMUR agent (e.g., an anti-NMU antibody)is co-administered with another agent, e.g., a second anti-NMU/NMURagent, or co-administered with another therapy for obesity and/ordiabetes. The terms “co-administration”, “co-administer”, and “incombination with” include the administration of two or more therapeuticagents (e.g., two or more anti-NMU/NMUR agents such as two differentanti-NMU antibodies, an anti-NMU antibody and an RNAi agent that targetsNMUR1, an anti-NMU/NMUR agent and an agent for treating diabetes, etc.)either simultaneously, concurrently or sequentially within no specifictime limits. In one embodiment, the agents are present in the cell or inthe subject's body (in their blood stream) at the same time or exerttheir biological or therapeutic effect at the same time. In oneembodiment, the therapeutic agents are in the same composition or unitdosage form. In other embodiments, the therapeutic agents are inseparate compositions or unit dosage forms. In certain embodiments, afirst agent can be administered prior to (e.g., minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours,96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks,or 12 weeks after) the administration of a second therapeutic agent.

In some cases, a subject an anti-NMU/NMUR agent (e.g., an anti-NMUantibody) (e.g., formulated as a pharmaceutical composition) isco-administered with a obesity and/or diabetes therapeutic drug, atherapeutic drug to treat a cancer such as PDAC, and the like. Suchadministration may involve concurrent (i.e. at the same time), prior, orsubsequent administration of the drug/antibody with respect to theadministration of an agent or agents of the disclosure. A person ofordinary skill in the art would have no difficulty determining theappropriate timing, sequence and dosages of administration forparticular drugs and compositions of the present disclosure.

An anti-NMU/NMUR agent (e.g., an anti-NMU antibody) need not be, but isoptionally formulated with one or more agents that potentiate activity,or that otherwise increase the therapeutic effect. These are generallyused in the same dosages and with administration routes as used hereinor from 1 to 99% of the employed dosages. In some embodiments, treatmentis accomplished by administering a combination (co-administration) of asubject anti-NMU/NMUR agent and an agent that treats obesity. In someembodiments, treatment is accomplished by administering a combination(co-administration) of a subject anti-NMU/NMUR agent and an agent thattreats diabetes. In some embodiments, treatment is accomplished byadministering a combination (co-administration) of a subjectanti-NMU/NMUR agent and an agent that treats a cancer such as PADC.Thus, also envisioned herein are compositions (and methods that use thecompositions) that include: (a) an anti-NMU/NMUR agent (e.g., ananti-NMU antibody); and (b) at least one of: (i) an agent used fortreatment of diabetes, (ii) an agent used for treatment of obesity, and(iii) an agent used for treatment of cancer, e.g., PDAC.

Delivery

An anti-NMU/NMUR agent (e.g., an anti-NMU antibody) can be administeredby any suitable means (e.g., systemic or local), including topical,oral, parenteral, intravenous, intrapulmonary, and intranasal.Parenteral infusions include intramuscular, intravenous (bollus or slowdrip), intraarterial, intraperitoneal, intrathecal or subcutaneousadministration. An anti-NMU/NMUR agent (e.g., an anti-NMU antibody) canbe administered in any manner which is medically acceptable. This mayinclude injections (e.g., by parenteral routes such as intravenous,intravascular, intraarterial, subcutaneous, intramuscular, intratumor,intraperitoneal, intraventricular, or intraepidural), or others as wellas oral, nasal, ophthalmic, rectal, or topical. Sustained releaseadministration is also specifically included in the disclosure, by suchmeans as depot injections or erodible implants.

As noted above, an anti-NMU/NMUR agent (e.g., an anti-NMU antibody) canbe formulated with a pharmaceutically acceptable carrier (one or moreorganic or inorganic ingredients, natural or synthetic, with which asubject agent is combined to facilitate its application). A suitablecarrier includes sterile saline although other aqueous and non-aqueousisotonic sterile solutions and sterile suspensions known to bepharmaceutically acceptable are known to those of ordinary skill in theart. An “effective amount” refers to that amount which is capable ofameliorating or delaying progression of the diseased, degenerative ordamaged condition. In some cases, an effective amount is an amount thatbrings about a rise in circulating insulin levels in the individual. Aneffective amount can be determined on an individual basis and will bebased, in part, on consideration of the symptoms to be treated andresults sought. An effective amount can be determined by one of ordinaryskill in the art employing such factors and using no more than routineexperimentation.

An anti-NMU/NMUR agent (e.g., an anti-NMU antibody) is oftenadministered as a pharmaceutical composition comprising an activetherapeutic agent and another pharmaceutically acceptable excipient. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

In some embodiments, pharmaceutical compositions can also include large,slowly metabolized macromolecules such as proteins, polysaccharides suchas chitosan, polylactic acids, polyglycolic acids and copolymers (suchas latex functionalized Sepharose™, agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins, peptides, and polysaccharides such as aminodextran, each ofwhich have multiple sites for the attachment of moieties. A carrier mayalso bear an anti-NMU/NMUR agent (e.g., an anti-NMU antibody) bynon-covalent associations, such as non-covalent bonding or byencapsulation. The nature of the carrier can be either soluble orinsoluble for purposes of the disclosure. Those skilled in the art willknow of other suitable carriers for binding anti-NMU/NMUR agents (e.g.,one or more anti-NMU antibodies), or will be able to ascertain such,using routine experimentation.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Compositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes,Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of thisinvention can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained or pulsatile release of the active ingredient. Thepharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the anti-NMU/NMUR agents (e.g., one or more anti-NMUantibodies) can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) or the LD₁₀₀ (the dose lethalto 100% of the population). The dose ratio between toxic and therapeuticeffect is the therapeutic index. The data obtained from these cellculture assays and animal studies can be used in further optimizingand/or defining a therapeutic dosage range and/or a sub-therapeuticdosage range (e.g., for use in humans). The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition.

In some cases, a method treating an individual (e.g., one who is obeseand/or has diabetes), includes, as described elsewhere herein,predicting whether an individual will benefit from such treatment (e.g.,measuring an expression level of NMU, such as NMU protein in abiological sample such as serum from the individual, and determiningwhether the individual will benefit, where those with an increased NMUlevel, e.g., relative to a reference value, will benefit).

Evaluation Steps (Verify/Evaluate/Monitor Steps)

In addition to increasing insulin output, administration of a subjectanti-NMU antibody can enhance output of crucial incretin hormones likeglucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP),further enhancing glucose regulation. Thus, in some cases, a subjectmethod is a method of increasing GLP-1 and/or GIP; decreasing glucagon;and/or decreasing glucose level(s).

In some cases, a subject method (e.g. a treatment method) includes,after administration of an anti-NMU/NMUR agent to an individual (e.g.,an anti-NMU antibody), a step of measuring one or more features of theindividual (e.g., to verify that the agent produces a desired outcome).Suitable features that can be measured include, but are not limited to:insulin level (e.g., an amount of circulating insulin); blood glucoselevel; an expression level of GLP-1 (e.g., GLP-1 protein) and/or GIP(e.g., GIP protein); glucagon level; glucose tolerance; body fat mass;patient weight; a response to an overnight fast; a meal tolerance test(MTT); and the like. In some cases, such measurements can be made aftera provocation (e.g., an overnight fast; a glucose challenge, a mealtolerance test (MTT), etc.).

In some cases, a subject method (e.g., a method of predicting, atreatment method, etc.) includes a step of measuring one or more of:insulin level (e.g., an amount of circulating insulin); blood glucoselevel; an expression level of GLP-1 (e.g., GLP-1 protein) and/or GIP(e.g., GIP protein); glucagon level; glucose tolerance; body fat mass;patient weight; a response to an overnight fast; a meal tolerance test(MTT); and the like. In some cases, such measuring can be used as anevaluation/monitoring step to test whether a treatment method (e.g., amethod that includes administration of an anti-NMU/NMUR agent such as ananti-NMU antibody or fragment thereof) has produced the desired outcome.In some cases, such measurements can be made after a provocation (e.g.,an overnight fast; a glucose challenge, a meal tolerance test (MTT),etc.).

In some cases, such measuring can be used as part of a prediction method(e.g., a method of predicting whether an individual will developDiabetes, PDAC, and/or pancreatitis; a method of identifying anindividual that would benefit from therapy with an anti-NMU/NMUR agent;etc.). For example, one can take into account an amount of measured NMUas well as an amount of measured insulin, glucagon, GLP-1, GIP, and/orglucose when formulating a prediction. In some cases, such measurementscan be made after a provocation (e.g., an overnight fast; a glucosechallenge, a meal tolerance test (MTT), etc.).

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

EXAMPLES Example 1: Two-Way ELISA

A two-way enzyme-linked immunosorption assay (ELISA) was built tomeasure either human or mouse neuromedin U (NMU) using two newly mademonoclonal antibodies (C578 and 2A16). Mouse hybridoma lines weregenerated that secrete monoclonal antibodies that bind human NMU. Theseantibodies were used to build the two-way ELISA, one for NMU capture anda separate one for detection. This assay permitted fast and sensitivedetection directly from human serum or plasma.

Human NMU is a 25 amino acid hormone encoded by the gene NMU andproduced from a processed pre-prohormone (Mitchell et al., Br JPharmacol. 2009 September; 158(1):87-103). NMU is produced in thegastrointestinal tract of humans and other mammals like mice. NMU has noknown covalent modifications other than C-terminal amidation (a commonfeature of circulating peptide hormones), and the newly generatedantibodies (C578 and 2A16) were generated against a bioactive form ofcirculating NMU. Two mouse hybridoma lines (C578 and 2A16) producingmonoclonal antibodies that bind NMU at picomolar affinity weregenerated, and CDR sequences were obtained from these cells (e.g., toproduce humanized versions of the antibodies) (See FIG. 10 for sequenceinformation). The anti-NMU antibodies were used to build a two-way ELISAthat can measure NMU in serum samples (e.g., in mouse serum and in humanserum).

NMU potently suppresses pancreatic islet secretion of insulin and alsosuppresses gastrointestinal secretion of the incretin hormones GLP-1 andGIP. NMU also stimulates secretion of the islet hormone glucagon.Insulin, incretins and glucagon are each fundamental regulators ofmetabolism in humans. Thus, identifying NMU functions in metabolism andgastrointestinal physiology was focused on. This has led to thediscovery that NMU levels are consistently elevated in metabolicdisorders like obesity and diabetes (e.g., see data presented in theother examples below). The two way-ELISA assay for measuring human NMUdisclosed herein has revealed increased circulating NMU levels inpathological states like obesity and pancreas cancer in humans (e.g.,see examples below). This two-way ELISA provides several clearadvantages over existing technology, which use a ‘one-way’ competitiveELISA requiring NMU binding to a single ‘capture’ antibody, competitionwith a prefabricated NMU standard linked to biotin, and subsequentdetection of biotin with avidin-conjugated peroxidase. The one-waycompetitive ELISA takes a longer assay time (>5 hours) and requiressample concentration steps that introduce significant variability.Moreover, the currently available assay fails to measure NMU in serumsamples. Thus, it has been (erroneously) reported previously that NMUdoes not circulate (Mitchell et al., Br J Pharmacol. 2009 September;158(1):87-103).

Obesity and Type 2 Diabetes Mellitus

Consistent with NMU signaling suppressing insulin output in vivo,compensatory increases of insulin and GLP-1 were found in mice withdiet-induced obesity (D10) or genetic insulin resistance (mutant db/dbmice), and were accompanied by reduced serum NMU levels. However, withadvancing age, these mice develop a well-recognized butpoorly-understood beta-cell failure, accompanied by reduced insulinsecretion, reduced serum insulin levels and impaired glucose control.These changes were strikingly accompanied by increased serum NMU levels.Glucose intolerance was eliminated and insulin output enhanced in theseolder obese mice with NMU antibody injection, or by genetic NMUsignaling blockade.

These findings identify obesity as a pathological state accompanied byNMU excess, are supported by the finding that serum NMU levels wereincreased in obese humans (FIG. 4A), and support the view that NMUantibodies can ameliorate metabolic consequences of obesity in vivo.Thus, NMU regulation may be impaired in subsets of obese humans, andelevated NMU levels are a biomarker of susceptibility to complicationsof obesity, including impaired glucose tolerance and frank diabetesmellitus. Moreover, these findings suggest that attenuation of NMUsignaling by antibody blockade in humans (e.g., obese humans) canameliorate or reverse complications of obesity including impairedinsulin output, hyperglycemia or frank diabetes mellitus.

Other common but poorly understood human disease states may reflect NMUexcess, including diabetes in lean subjects (as observed in Asians, theelderly, and chronic pancreatitis), or refractory hyperglycemia afterbariatric surgery. Thus, prospective measures of NMU levels andregulation may permit a new kind of disease stratification, and identifypatient subsets who may be responsive to targeted NMU signalingattenuation using agents like NMU-neutralizing antibodies.

Antibody-based reduction of NMU signaling has possible therapeuticapplications in a potentially large group of subjects with excessivesystemic NMU signaling or levels. Our studies show NMU elevation in (1)patients with pre-diabetes states like obesity or with establisheddiabetes, (2) patients recognized to have ‘lean’ diabetes, includingsubjects with advanced age (>60 y.o.), exocrine pancreas diseases likecystic fibrosis, familial or idiopathic pancreatitis, or the recentlyrecognized ‘type 3c’ diabetes mellitus, (3) patients with advancedcancer states, like late stage pancreatic cancer and a debilitatingcomplication called cancer cachexia.

Chronic Pancreatitis, Type 3c Diabetes Mellitus and Cancer

Epidemiological studies show that chronic pancreatitis is linked toincreased risk of diabetes mellitus and pancreas cancer. Using archivedhuman samples, we have now discovered that NMU is mis-produced in areasof cell metaplasia in a subset of human pancreatitis (without detectableadenocarcinoma), and that serum levels of NMU are elevated inpancreatitis and significantly further elevated in PDAC (e.g., seeexamples below). Thus, NMU production can be used as a biological markerof cell metaplasia (pre-cancer) and diabetes risk in the pancreas.Because NMU both circulates and has biological activity (suppressesinsulin and incretin secretion, promotes glucagon secretion), NMU excessin pancreatitis, or early stage pancreas cancer could promote a form ofdiabetes called ‘type 3c.’ Thus, NMU may be a long-sought molecular linkbetween diabetes and pancreas cancer.

Two-Way ELISA

Using the two-way ELISA disclosed herein, mouse Nmu and human NMU weredetected in a range of from 0.1 to 20 ng/mL. Serum NMU levels wereincreased in humans with increasing BMI (FIG. 4A), an association thatis statistically significant. In mice and humans without known disease,an approximately 20-fold range of NMU levels in serum was observed. Themechanisms underlying increased serum NMU levels in obesity remainunclear. This may reflect a response to suppress feeding (e.g., NMUover-expression in mice can induce hypophagia). Alternately, this mayreflect pathological mechanisms leading to inappropriate NMU secretion.

Example 2

FIG. 1A-1E: Physiological Dynamics of Human and Mouse NMU In VivoRevealed by NMU ELISA Assays.

Neuromedin U (NMU) is the mammalian ortholog of limostatin, astarvation-induced peptide hormone identified in the fruit flyDrosophila. NMU is an enteroendocrine hormone that regulates humaninsulin output by acting through its receptor, NMUR1, expressed onpancreatic beta-cells (Alfa et al., Cell Metab. 2015 Feb. 3;21(2):323-33). Consistent with this view, while circulating glucose(FIG. 1A) and insulin (FIG. 1B) levels decrease during prolonged fastingin a human subject, NMU signaling is increased as reflected by elevationof circulating NMU levels in human (FIG. 1C) and mice (FIG. 1D) fastedfor up to 72 hours. Loss of peripheral NMU signaling in mice lackingNMUR1 induced an additional accumulation of NMU in plasma of mice fastedfor 48 and 72 hours (FIG. 1E). Thus, NMU signaling in fasting isconserved. In addition, these data demonstrate that the two-way ELISAassay (and kit) described herein is a new tool that can measure NMU invivo in the serum of humans and mice. The two-way ELISA assay used inthe experiments of this figure included the antibodies C578 and 2A16(both of which are described herein).

FIG. 2A-2F: Evidence that NMU Suppresses Post-Prandial Output in Humansand Mice.

Human serum insulin levels rise rapidly by 30 minutes after oral glucosechallenge, then fall over the ensuing 150 minutes (FIG. 2A), leading toglucose disposal. By contrast human NMU levels rapidly decreased by 30minutes after oral glucose challenge, then gradually increased over thenext 150 minutes (FIG. 2B), demonstrating reciprocal regulation of thesetwo hormones upon enteral nutrient intake. In mice, circulating NMUlevels were increased within 30 minutes after a meal tolerance test(MTT) and reached a maximum peak at 120 minutes. Then the NMU levelsremained high in a plateau and returned back to basal levels within 5 hpost meal challenge (FIG. 2C). It was confirmed that 30 minutes after anoral bolus of glucose, plasma NMU levels increase (FIG. 2D) whereas anintraperitoneal bolus of glucose did not trigger serum NMU dynamics,demonstrating that the secretion of decretin hormone is regulated bynutrient sensing mechanisms in gastrointestinal tract (FIG. 2E). Micewere subjected to serial oral glucose challenges leading to expecteddynamic insulinemic and glycemic responses. NMU levels weresignificantly higher in the systemic circulation of mice at 30 minutesafter the first oral glucose challenged and stayed in a plateau. Uponsecond glucose challenge at 60 minutes, rapid reduction of NMU levelswas observed in plasma within 2 minutes followed by a sustained NMUincrease for at least 4 hours (FIG. 2F). Thus, circulating NMU levelsare acutely regulated in the post-prandial setting. In both humans andmice, oral glucose challenge can induce an initial fall then a sustainedrise of NMU serum levels. These excursions are reciprocal to the risethen fall of serum insulin.

FIG. 3A-3F: In Vivo Loss of NMUR1 Signaling Pathway Improves GlucoseClearance During Prolonged Fasting.

After prolonged fasting, refeeding has been shown to lead to starvationdiabetes as was observed in fasted humans (FIG. 3A-B) and mice (FIG.3C-D) challenged with glucose or meal. In mice and humans 72 hourfasting induced a right shift of insulin excursion and significanthyperglycemia (FIG. 3A-D). To determine the significance of NMU duringfasting, NMUR1 mutant mice were generated which lack NMU receptor 1.Fasted NMUR1−/− mutant mice challenged with an oral glucose bolus,displayed a higher insulin response and a better glucose clearance (FIG.3E-F). Thus, deletion of NMUR1 signaling prevented starvation diabetes.Thus, in vivo attenuation of NMU signaling improved glucose toleranceand insulin output to prevent diabetes.

FIG. 4A-4C: NMU Dynamics in the Pathological Setting of Obesity.

Obese and pre-diabetic human subjects often develop relative insulindeficiency and glucose intolerance. Given the data presented here frommice and humans that correlate NMU with reduced insulin excursion, itwas hypothesized that NMU might be dysregulated in obesity andpre-diabetic states associated with glucose intolerance. Serum NMUlevels were increased in obese humans (FIG. 4A, BMI>30, R²=0.41,P<0.001). Diet-induced obesity from high fat diet (HFD) challenge inmice is accompanied by a compensatory hyperinsulinemia lasting 6-10weeks. In that period, circulating NMU levels were reduced (FIG. 4B).After 12-24 weeks of HFD, plasma NMU levels increased significantlywhile basal insulin levels were no longer high (FIG. 4C) and wildtypeanimals displayed impaired glucose clearance in response to an oralglucose challenge.

FIG. 5A-5C: Loss of NMU Signaling In Vivo Improves Metabolism inObesity.

Null mutant mice lacking NMUR1 were more prone to gain weight when fed ahigh-fat diet compared to wild type mice (FIG. 5A). However, NMUR1genetic deletion in diet-induced obese mice improved insulin excursionand glucose tolerance in response to a glucose challenge (FIG. 5B-C).Thus in obesity states, NMU signaling attenuation improves insulinoutput and glucose control.

FIG. 6A-6E: NMU is a Novel Regulator of GLP1 and GIP.

The increased circulating levels of NMU observed in mice during fastingwere reproduced with a single intraperitoneal dose of NMU (FIG. 6A).Enhanced NMU reduced basal levels of circulating incretin levels, GLP1and GIP (FIG. 6B-C). GLP1 and GIP are produced and secreted from theintestinal tract, including the ileum. Using a mouse ileum culturesystem, glucose-induced secretion of GLP1 and GIP were suppressed by NMU(FIG. 6D-E).

FIG. 7A-7D: NMU Signaling Attenuation Enhances Incretin Output.

In NmuR1 null mutant mice, NMU inhibition of glucose-induced GLP1 andGIP output was lost in vivo (FIG. 7A-7B: compare to FIG. 6B-FIG. 6C). Incultured ileum from NmuR1 null mutant mice, NMU inhibition ofglucose-induced GLP1 and GIP output was lost (FIG. 7C-7D; compare toFIG. 6D-6E).

FIG. 8A-8B: NMU Signaling Decreases Insulin and Increase Glucagon Outputin Vivo.

Increased plasma NMU in mice lowered basal insulinemia (FIG. 8A) andenhanced glucagonemia (FIG. 8B).

FIG. 9A-9B: Anti-NMU Antibody Therapy Promotes Insulin Secretion andEliminates NMU-Induced Glucose Intolerance.

A single injection of NMU impaired insulin excursion in response to ameal challenge and led to hyperglycemia over 120 minutes. Co-injectionwith an anti-NMU monoclonal antibody that binds to a region common tomouse and human NMU (antibody C578, see FIG. 10) neutralized circulatingNMU levels, restored insulin secretion (FIG. 9A) and improved glucoseclearance (FIG. 9B).

FIG. 10.

CDR sequences were obtained from both light and heavy chains of newlygenerated anti-NMU antibodies (2A16, C578) that bind to human NMU (C578binds to both human and mouse NMU).

Example 3 Neuromedin Signaling Basis of Diabetes in Chronic Pancreatitisand Pancreatic Ductal Adenocarcinoma

Investigating the Neuromedin Signaling Basis of Diabetes in ChronicPancreatitis and Pancreatic Ductal Adenocarcinoma

Neuromedin U (NMU) is a peptide hormone produced by enteroendocrinecells and

CNS neurons. In pre-clinical studies, NMU is a potent suppressor ofinsulin secretion by human islets. NMU also suppresses secretion of theincretin hormone GLP-1. The data and investigations provided hereindemonstrate that NMU is mis-expressed in the pancreas in humans withchronic pancreatitis, and established pancreatic ductal adenocarcinoma,and that serum NMU levels are elevated in subsets of subjects with PDACor pancreatitis. Thus, ectopic pancreatic NMU may promote pancreatogenicdiabetes mellitus (T3cDM) and serum NMU levels can be a biomarker ofchronic pancreatitis, pancreatic metaplasia or neoplasia.

Nmu is a Hormone that Suppresses Insulin Secretion by Pancreatic Islets

A secreted hormone, Limostatin (Lst), suppresses insulin secretion andproduction following starvation in Drosophila (Alfa et. al., Cell Metab.2015 Feb. 3; 21(2):323-33). Lst is produced and secreted by fly corporacardiaca cells, best known as cells that also secrete adipokinetichormone (AKH), a functional orthologues of glucagon. Many neuropeptidessignal through G protein-coupled receptors. All known orphan GPCRs werescreened in Drosophila and Computed Gene #9918 (CG9918) was identifiedas a GPCR expressed in IPCs that negatively regulates insulin expressionand secretion in the adult fly. Pharmacogenetic findings indicated thatLst regulates insulin secretion directly in IPCs, and support the viewthat CG9918 encodes a Lst receptor. The transmembrane domains encoded bythe fly Lst receptor have highest sequence homology to the mammalianneuromedin U receptor 1 (NmuR1). Nmu encodes a pre-prohormone expressedin the brain (including hypothalamic nuclei) and in peripheral organs,including abundant expression in gastrointestinal organs. Thus, mouseNMU and human NMU production in gastrointestinal organs was assessed. Inhumans, NMU immunoreactivity was localized to scattered enteroendocrinecells that co-expressed Chromogranin B and were distributed in thegastrointestinal tract, prominently in stomach pyloric mucosa, duodenumand ileum (FIG. 11A-11B). Consistent with these results, Nmu mRNAexpression was detected prominently in human stomach, duodenum, jejunum,ileum and colon. A similar distribution of Nmu⁺ cells was detected inthe adult mouse gastrointestinal tract. In the stomach, the greatestnumber of Nmu⁺ cells was found in the cardia and greater curvature.Based on the intestinal segment lengths, the mouse and human ileumcontains the greatest number of Nmu⁺ cells. By contrast there was littleto no detectable NMU production in mouse or human pancreas.

In humans and other mammals, peripheral effects of Nmu are mediated byNmuR1, while NmuR2 is primarily expressed in the CNS. mRNA encodingNmuR1 but not NmuR2 was readily detected by qPCR and in situhybridization in isolated human and mouse islet β-cells. NmuR1production was restricted to insulin⁺β cells (FIG. 12A). Little to nosignal was detected in glucagon⁺α cells, somatostatin 6 cells orexocrine ducts and acinar cells. To test directly if NMU can suppressinsulin secretion, islets were isolated and glucose-stimulated insulinsecretion (GSIS) was assessed at a concentration of NMU reported toelicit physiological responses (Jones et. al., Regul Pept. 2006 Sep. 11;136(1-3):109-16). Human NMU potently suppressed glucose-stimulatedinsulin secretion from human islets in static batch culture assays (FIG.12B-12C) and islet perifusion experiments. An NMU R165W allele thatencodes a mutant peptide was previously found to co-segregate in anautosomal dominant pattern with early-onset obesity andhyperinsulinemia. In human islet perifusion assays, the R165W NMUvariant failed to suppress insulin secretion compared to wild-type NMU.These data suggest that the human NMU R165W mutation represents ahypomorphic loss-of-function allele, and that impaired regulation ofinsulin secretion by NMU underlies metabolic changes in carriers of thisallele. Collectively, the data presented here shows that NMU is producedin the gastrointestinal tract (but not in the pancreas) and thatnanomolar levels of NMU strongly inhibit insulin secretion by β-cells inmouse and human islets.

Nmu Infusion In Vivo Regulates Insulin, GLP-1 and Glucagon Output, andGlucose Tolerance

The ability to measure serum NMU protein in mice (e.g., using thetwo-way ELISA disclosed in herein) permitted studies to investigate theimpact of in vivo NMU infusion on crucial regulators of metabolism likeinsulin and glucagon. It was confirmed that a simple schedule of dailyinfusion by intraperitoneal injection led to a two-fold increase of meanserum NMU levels (FIG. 13A). Serum insulin and GLP-1 levels werereduced, while glucagon levels were increased (FIG. 13B-13D). Consistentwith these findings, reduced insulin secretion and impaired glucosetolerance were detected after oral glucose tolerance testing in miceinfused with NMU (compared to vehicle-infused controls; FIG. 13E-13F).Thus, acute NMU infusion leading to a doubling of serum concentrationwas sufficient to evoke changes of Insulin, GLP-1 and Glucagon thatpromote a diabetes-like state. Based on these findings, it washypothesized that states of relative NMU excess in humans may affectmultiple hormones, including insulin, GLP-1 and glucagon.

NMU Protein is Mis-Expressed in Human Chronic Pancreatitis and PDAC

NMU protein is not detectable in human pancreatic duct, acinar or isletcells of subjects without known pancreatic diseases, and usingimmunohistology no NMU was detected in islets, ducts or acinar cells ofpancreas tissue from such donors (FIG. 14A). In the spectrum of injuryand inflammation leading to metaplasia and progression to neoplasia invisceral organs, ectopic expression of hormones can lead to‘para-neoplastic’ syndromes, like in small cell lung cancerpathogenesis. To investigate the possibility of using NMU as a biomarkerof chronic pancreas injury, the possibility that NMU may bemis-expressed by pancreatic cells in chronic pancreatitis or pancreaticductal adenocarcinoma was assessed. In 2/3 cases of chronicpancreatitis, immunohistology studies revealed ectopic NMU production inductal epithelium (FIG. 14B). Likewise in 2/2 cases of PDAC, NMUexpression in duct-like cells was observed (FIG. 14C-14D). By contrast,in areas without evidence of tumor infiltration in both PDAC cases, orin 2 cases of acute pancreatitis, NMU production in the pancreas was notobserved.

NMU is not normally detected in human islets. Surprisingly, in a subsetof islets adjacent to areas of obvious neoplasia in both PDAC cases, NMUimmunostaining was observed in islet cells. Co-labelling with antibodiesspecific for NMU, Insulin and Glucagon demonstrated that Glucagon⁺ cells(but not Insulin⁺ cells) mis-expressed NMU (FIG. 15). These findingssuggest that subsets of human islets may be influenced by PDAC, leadingto ectopic production of NMU in islets. Together, the data areconsistent with pancreatogenic NMU in chronic pancreatitis or PDAC beinga biomarker and promoter of disease progression.

Meta-Analysis of NMU mRNA Expression in PDAC

To assess whether NMU mRNA levels are elevated in PDAC existinggenome-scale expression profiles from human PDAC were analyzed. Briefly,meta-analysis approaches were used to assess data sets from a total of264 PDAC and 91 normal expression profiles normalized using gcRMA(Khatri et al, Ann Intern Med. 2013 Jan. 1; 158(1):35-46; Chen et al.,Cancer Res. 2014 May 15; 74(10):2892-902). Hedges' adjusted g test wasused for each gene to combined effect sizes (black squares) from eachdataset into a pooled effect size (yellow diamond) to estimate theamount of change in expression across all datasets. A pooled effect sizeand standard error were obtained by combining effect sizes from eachdataset using the random effects inverse-variance technique. P value wascomputed from the z-statistics, and adjusted using the BenjaminiHochberg method. This revealed a significant increase of mRNA encodingNMU in this sampling (P<10⁻⁷), but not mRNA encoding neuromedin B (NMB:FIG. 16). Thus, a set of independent data supports the hypothesis thatpancreatic NMU expression is abnormally increased in PDAC.

NMU ELISAs Reveal Elevated NMU Levels in PDAC Associated with ReducedBMI and Cachexia

ELISAs for mouse and human NMU are sold commercially, but havesignificant limitations. In fact, prior reports claim that NMU wasundetectable in rodent serum, leading to the incorrect conclusion thatNMU chiefly functions as a paracrine signal (Mitchell et al., Br JPharmacol. 2009 September; 158(1):87-103). In the ‘competitive’ one-wayassays, blood and other biological samples require purification andlyophylization of >1 milliliter of serum to reduce the impact ofendogenous biotinylated macromolecules, and this concentration stepintroduces significant variation or irreproducibility. In another set of‘two-way’ ELISAs, the immunogen was pre-proNMU and these kits do notdetect processed bioactive human NMU in serum. To overcome this, a new2-way ELISA assay was built with novel monoclonal antibodies. This hassignificantly improved assay reproducibility, and permits use of 10microliters of serum, without a concentration step. Mouse NMU proteinand human NMU protein can now be detected at 0.1 to 20 ng/mL (FIG. 17).Detection of NMU in human or mouse serum does not require addition ofprotease inhibitors as required for stabilizing GLP-1.

With this new human ELISA serum NMU was measured in control subjectswithout cancer or with advanced pancreatic ductal adenocarcinoma (PDAC).In controls without known pancreatic disease or T2DM (type 2 diabetes),NMU levels increased with BMI, an association that was statisticallysignificant (FIG. 17: Spearmann Rank=0.69). By contrast two strikingfeatures of NMU levels in PDAC patients were observed. First, mean serumNMU levels were elevated about two-fold compared to controls (FIG. 17:note the differing Y-axis scales). This finding is consistent with theanalysis that revealed increased pancreatic NMU mRNA expression in PDAC,and indicates that this ectopic pancreatic expression may result inelevated serum NMU levels. In turn, this supports the view that thepara-neoplastic effects of NMU may be both paracrine and endocrine.Excluded from this analysis of PDAC are two subjects in whom very highlevels of NMU were observed, including one subject with PDAC anddiabetes mellitus whose NMU level was 19.5 ng/mL (NMU level >3 SD abovemean). Although these differences are not yet statistically significant,this likely reflects the relatively small number of patients analyzed inthe preliminary studies. Second, unlike in controls, a striking increaseof NMU levels was observed in patients with low BMI, an association thatwas statistically significant (FIG. 17; Spearmann Rank=−0.83;P=0.00015). In the subset of patients with highest NMU and lowest BMI,there was clinical evidence of tumor cachexia. In prior studies oftransgenic mice, systemic NMU overproduction was found to reduce bodymass, decrease adiposity and food intake, resulting in lean mice. Thus,NMU over-production can produce states mimicking pre-cachexia. Using thesystems discussed above, it was shown that doubling of serum NMU levelsis sufficient to impair insulin and incretin output, and disruptedglucose regulation (e.g., see FIG. 13 above). It is suggested here thatectopic pancreatic NMU is a para-neoplastic factor that can promotediabetes, cachexia and other metabolic alterations.

Evidence for Increased Serum NMU Levels in Subjects with ChronicPancreatitis

The studies presented here related to gene expression and NMU proteinproduction in pancreatitis suggested that serum levels of NMU might bedetectably increased in this setting. To test this, feasibility studiesof patients with acute and chronic pancreatitis were performed. Cleartrends were observed indicating that the mean serum levels of NMU wereincreased in subjects with chronic pancreatitis (and pancreatic cancer),compared to those with acute pancreatitis or control subjects withoutexocrine pancreas disease (FIG. 18). These findings highlight thedynamic range of the two-way ELISA serum assay disclosed herein, andindicate that the assay can identify elevated NMU levels in pancreatitisand PDAC.

An interesting link of these findings comes from the observation thatproduction of cytokines like IL-6 in macrophages is regulated by NMU.Thus, a pro-inflammatory role for NMU is implicated via its ability toinduce synthesis and release of Th2 cytokines. Studies in mice suggestthat Th2 cytokines and IL-4R□ signaling activates pancreatic stellatecells, promoting the characteristic fibrogenesis observed in chronicpancreatitis progression. These results are substantiated by findingsfrom human ex-vivo co-cultures of PSCs and macrophages. Thus ectopicexpression of NMU in chronic pancreatitis and PDAC (FIG. 14, FIG. 15)could amplify Th2 signaling to perpetuate progression of disease.

The investigations of NMU signaling presented herein suggest (1) thatcirculating NMU regulates a physiological signaling pathway thatcontrols insulin and GLP-1 output in vivo (FIG. 19A), and (2) thispathway is perturbed in common chronic exocrine pancreas diseases likepancreatitis and pancreas adenocarcinoma. In these diseases, the datashow that NMU expression (mRNA and protein production) are abnormallyincreased in the human pancreas, where NMU is not normally produced(FIG. 19B). Sites of ectopic NMU production may include metaplasticducts, neoplastic cells and islet cells. Based on the finding that NMUcirculates, and is measurable by the two-way ELISA disclosed herein,elevated NMU levels in chronic pancreatitis and PDAC can be detected insubsets of patients, and NMU likely suppresses output of insulin andincretins like GLP-1, leading to dysregulated glucose homeostasis. Thus,pancreatogenic NMU may reflect ongoing cellular metaplasia or neoplasiain these diseases and thereby promote phenotypes found in pancreatogenicdiabetes, including relative hypoinsulinemia, hypoincretinemia, andimpaired glucose regulation.

Example 4

Mouse Nmu is produced in enteroendocrine cells in the stomach, duodenum,ileum and colon, while NmuR1 is expressed in islet β-cells. Based on thefindings presented in this disclosure, we suggest that enteroendocrineNmu serves as a crucial nexus between nutrient status, thegastrointestinal tract, and islet hormones to control metabolism. Wesuggested that Nmu signaling from gut to pancreas islets constitutes anew, physiologically important inter-organ signaling axis regulatingmammalian metabolism.

Nmu is produced both in the CNS and peripheral tissues, particularly inthe gastrointestinal tract. Nmu inhibits pancreatic insulin secretion.Nmu receptor1 (NmuR1) is a G protein-coupled receptor expressed in mouseand human islet β-cells. Factors that regulate β-cell insulin secretion,including the hormones somatostatin and galanin, and pertussis toxin,require the inhibitory G-protein G_(o)2 for proper secretory regulation.Mutations in human NMU are linked to hyperinsulinemia and obesity.

Starvation and feeding are potent selective forces, and hormonalresponses to maintain metabolic balance in the face of nutrientrestriction or abundance appear to be highly conserved in all animals.In mammals, powerful mechanisms potentiate insulin-secreting cellresponses to the incoming glucose load before glucose levels rise. Forexample, incretin hormones such as glucagon-like peptide-1 (GLP-1) aresecreted by enteroendocrine cells following a meal, and enhanceglucose-stimulated insulin production and secretion from pancreatic βcells.

Prior to the work described in this disclosure, an enteroendocrinehormone induced by fasting that decreases insulin output (a “decretin”)had not been identified. Loss of such a hormone has been postulated asone basis for the rapid resolution of diabetes phenotypes in obesepatients following gastric bypass surgery (Rubino et al 2009), anincreasingly common intervention.

Nmu is Functional and Inhibits Insulin Secretion by Pancreatic Islets

The transmembrane domains encoded by CG9918 have highest sequencehomology to the mammalian Neuromedin U receptor 1 (NmuR1). Nmu encodes apre-prohormone expressed in the brain (including hypothalamic nuclei)and in peripheral organs, including abundant expression ingastrointestinal organs. In mice, Nmu is a 23 amino acid peptide(Nmu-23); in human NMU is 25 amino acids (NMU-25). Mouse Nmu and humanNMU production was assessed in gastrointestinal organs. In humans, NMUimmunoreactivity was localized to scattered enteroendocrine cells thatco-expressed Chromogranin B with a typical ‘open type’ morphology, andwere distributed in the gastrointestinal tract, prominently in stomachpyloric mucosa, duodenum and ileum (FIG. 20A-B). Consistent with theseresults, NMU mRNA expression was detected prominently in human stomach,duodenum, jejunum, ileum and colon. To investigate mouse intestinal Nmucells, and understand mechanisms regulating Nmu production and secretionfrom enteroendocrine cells, mice were obtained that harbor a BACtransgene encoding an eGFP transgene ‘knocked-in’ to the Nmu locus. Inadult mice, Nmu-eGFP⁺ cells were readily detected by microscopy instomach, duodenum, ileum, jejunum and colon (FIG. 20B). Standard methodswere used to isolate and purify Nmu-eGFP⁺ cells by fluorescenceactivated cell sorting (FACS). FACS isolation of Nmu⁺ cells from stomachand small intestines yielded ˜50,000 Nmu⁺ cells per adult mouse, similarto the number of pancreatic islet α-cells. RNA-Seq analysis of duodenaland ileal Nmu-eGFP⁺ cells (FIG. 20C) revealed co-expression of Nmu withenteroendocrine markers like ChgA, ChgB, Pcsk1, Cpe, NeuroD1, Lmx1a andNkx2.2: none of these markers was detected in purified Nmu^(neg) cells.Nmu-eGFP⁺ cell expression was not detected for preproglucagon, GIP,Somatostatin, Motilin, Ghrelin or Ins15. Thus, Nmu⁺ cells appear to bedistinct from Nmu^(neg) cells, and from multiple known enteroendocrinecell types, including L-, K-, M- and D-cells. Comparison to mouse isletcell gene signatures by Pearson analysis revealed, as expected, thatNmu⁺ cells were distinct from Nmu^(neg) cells and similar to isletcells, especially δ- and α-cells (FIG. 20C). Like other endocrine cellsthat depolarize in response to secretogogues, Nmu⁺ cells expressed mRNAsencoding voltage-gated Ca²⁺ channels like Cacna1a, voltage-gated sodiumchannels like Scn3a, and the glucose transporter Slc2a1 (Glut1).Consistent with these findings, an established primary ileal cultureassay was used, and increased Nmu release was detected after exposure to20 mM potassium chloride. FIG. 20: Human and mouse NMU⁺ cells. (A)vl=mucosal villus, gl=gland.

In humans and other mammals, peripheral effects of Nmu are mediated byNmuR1, while NmuR2 is primarily expressed in the CNS. mRNA encodingNmuR1 but not NmuR2 is readily detected by RNA-Seq, qPCR and in situhybridization in isolated human and mouse islet β-cells, albeit at lowlevels (FIG. 21A). Little to no NmuR1 expression was detected inglucagon⁺ a, cells (FIG. 21B), somatostatin⁺δ cells or exocrine ductsand acinar cells. To test if NMU can suppress insulin secretion, humanislets were isolated and glucose-stimulated insulin secretion at 50-100nM, a concentration of NMU reported to elicit physiological responses,was assessed. Human NMU potently suppressed glucose-stimulated insulinsecretion from human islets in perifusion experiments (FIG. 21C).Similar effects were observed with mouse Nmu and purified mouse islets(FIG. 25). Collectively, the work disclosed herein show that NMU isproduced in the gastrointestinal tract (but not islets), and thatnanomolar levels of NMU strongly inhibit insulin secretion by β-cells inmouse and human islets. FIG. 21: (A-B) Human NMUR1 is expressed in isletβ-cells (A) but not in α-cells (B) or δ-cells. (C) Human isletperifusion shows NMU-25 suppresses insulin secretion.

Commercially-available ELISA assays have not previously achievedreliable measures of circulating NMU in mouse or human serum; hence itwas not clear that NMU was a bona fide endocrine hormone. To addressthis deficit, new 2-way ELISAs with newly generated monoclonalantibodies were built to measure NMU in these species. This new setupallows the use only 1-2 microliters of serum, without ahydrophobic-resin column concentration step. The new ELISAs detect mouseand human NMU in a working range spanning 0.2 to 900 ng/mL (FIG. 22).Nmu is detectable in mouse serum during ad libitum feeding, and Nmulevels increase after 48-72 hours fasting (FIG. 22A: n=3). Insulinlevels decline throughout this period, reflecting regulation both bynutrient restriction and circulating factors. Circulating Nmu in fastedmice fell to baseline levels within one hour after enteral feeding butnot after intraperitoneal glucose injection. Similar changes areobserved in human volunteers fasted 72 hours (FIG. 22B: n=2). Thus, NMUlevels increased during fasting in mice and humans. Together, thesefindings further support the concept that Nmu is a mammalian decretin.

To assess whether human serum NMU levels can be detected in more commonphysiological settings, serum NMU was measured in human subjectsstratified by insulin suppression testing. Age- and BMI-matched lean,non-diabetic women were stratified into insulin-resistant (IR: n=14) andinsulin-sensitive (IS: n=7) groups on the basis of their steady-stateplasma glucose (SSPG) concentration following a 180-minute infusion ofoctreotide, exogenous insulin, and glucose. Significant elevation ofaverage serum NMU was observed in the IR group (FIG. 22C: 5.0 vs. 20,P=0.019). Thus, this work reveals increased circulating NMU levels inhumans with impaired metabolism.

Nmu Infusion In Vivo Regulates Insulin and Glucagon Output, and GlucoseTolerance

Identification of the normal range of serum NMU levels in mice guidedstudies to investigate the impact of physiologically-relevant increasesof Nmu-23 after in vivo infusion (n=4). A doubling of NMU levels wasdetected within 10 minutes after IP infusion in 10 week-old B6J malemice fasted 12 hours (FIG. 23A). Serum insulin levels were reduced,while glucagon levels were doubled (FIG. 23B-C) after Nmu injection. Theregulation of glucagon by Nmu is likely to be indirect, since NmuR1 orNmuR2 expression has not been detected in mouse or human α-cells.Consistent with these findings, insulin secretion was reduced andglucose tolerance was impaired after oral glucose tolerance testing inmice infused with Nmu (compared to vehicle-infused controls; FIG. 27C).Relative hyperglucagonemia from Nmu infusion could enhance hepaticglucose production. To investigate this, hyper-insulinemic clamp studieswere performed. Compared to 10 week-old B6J male controls (n=7),isogenic mice infused with mouse Nmu-23 (0.3 mg/kg/hour; n=9) requiredreduced glucose infusion rate (GIR), and had reduced insulin-sensitivesuppression of hepatic glucose production (HGP), consistent withrelative hyperglucagonemia (FIG. 23D-F). FIG. 23: *P<0.05

Efficient Genetic Transduction and Functional Assessment of PrimaryHuman Islet Cells

Transgenes were expressed in human primary islet cells, by dispersingislets to single cell suspensions, and then reaggregated into clusters.Islet cell clusters after re-aggregation (also called ‘pseudoislets’)remained responsive to glucose and other secretion signals like IBMX.After transplantation in mice, pseudoislets vascularized and achievedregulated insulin secretion (FIG. 24). Lentiviral vectors were used toinfect human islet cells, to achieve expression of transgene-encodedproducts like nuclear Green Fluorescent Protein (nGFP). Less than 10% ofislet cells were transfected when intact cultured islets were infected.By contrast, nGFP production was detected in 75% of islet cells afterdispersion, lentiviral infection and re-aggregation (FIG. 24A).Immuno-panning and magnetic bead-based separation was used to purifydispersed β-cells and non-β-cells. This strategy achieved 140-foldpurification and >95% purity of CD26^(neg) hPI2⁺β-cells (FIG. 24A). Thispurification step allowed the targeting of primary human β-cells orα-cells using lentivirus, prior to reaggregation in pseudoislets. Afterlentiviral transduction and reaggregation, human pseudoislet insulinsecretion in vitro remained regulated (FIG. 24B).

This system was used to mis-express factors like SIX3, a homeodomaintranscription factor related to the Drosophila factor sine oculis.Remarkably, SIX3 expression in 2-year-old human pseudoislets enhancedbasal and glucose-stimulated insulin secretion (FIG. 24C-D). Afterpseudoislet transplantation in immuno-compromised mice, insulinsecretion after glucose challenge also remains regulated and robust,comparable to secretion from matched human islet controls (FIG. 24E).Consequently, glucose tolerance was improved similarly by transplantedcontrol islets or pseudoislets compared to sham-transplanted controls(FIG. 24F).

NMU Suppression of Insulin Secretion is Eliminated by PTX

Mouse islets exposed to pertussis toxin (PTX) had robust increases ofinsulin secretion, both at basal and increased glucose concentrations(FIG. 26), an effect thought to reflect PTX-induced degradation of theinhibitory G-protein G_(o)2. Importantly, PTX-treated mouse isletsbecome insensitive to mouse Nmu mediated suppression of insulinsecretion (FIG. 26). This supports the view that Nmu signaling requiresG_(o)2 activity, FIG. 27: Batch assay with mouse islets.

Reconstituting Starvation Diabetes in Mice with Nmu Infusion

To identify effects of Nmu elevation on metabolism, glycemic and insulinregulation were studied in 10 week-old B6J mice with elevated serum Nmuafter fasting. After intraperitoneal glucose tolerance testing (IPGTT)male or female mice fasted 72 hours (except for ad libitum water) hadprolonged hyperglycemia compared to matched control mice with IPGTTafter 24 hr. fasting (FIG. 27A-B) and blunted serum insulin levels 5minutes (0.16±0.1 vs 0.5±0.2 ng/ml, P<0.05: n=7 male mice) and 15minutes after glucose challenge. Thus, starvation provoked hallmarkfeatures of ‘starvation diabetes’. FIG. 28: Data for male mice shown.

Starvation provokes complex cellular and metabolic adaptations includingmultiple hormone responses. The doubling of circulating Nmu levels instarvation (FIG. 22A) suggested that Nmu might be sufficient to suppressinsulin output in vivo. To test this possibility without the complexityof starvation responses, mice were injected intraperitonally with NMUafter 12 hour fasting and insulin and glucose responses were measured.Serum NM levels rose 5 minutes after intraperitoneal injection andremained two-fold greater than (basal) fasting levels at 60 minutes(FIG. 27C), recapitulating the degree of Nmu increase seen after 72 hr.fasting in mice. Oral glucose challenge after Nmu injection in wildtypemice resulted in marked hyperglycemia (FIG. 27D) accompanied bysuppression of insulin levels. Thus, acute Nmu injection reconstitutedhallmark features of starvation diabetes.

What is claimed is:
 1. A method of increasing circulating insulin in anindividual in need thereof, the method comprising: administering to theindividual, an anti-neuromedin U (anti-NMU) antibody or antigen bindingregion thereof that reduces binding between NMU and NMUR1, at a doseeffective to increase the amount of circulating insulin in theindividual, wherein the anti-NMU antibody or antigen binding regionthereof comprises: (i) a light chain comprising a CDR-L1, CDR-L2, andCDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs:18-20, respectively, or SEQ ID NOs: 2-4, respectively; and/or (ii) aheavy chain comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the aminoacid sequences set forth in SEQ ID NOs: 26-28, respectively, or SEQ IDNOs: 10-12, respectively.
 2. The method according to claim 1, whereinthe individual has diabetes or is suspected of having an increased riskof developing diabetes.
 3. The method according to claim 1, wherein theindividual has a disease selected from: cystic fibrosis, familialpancreatitis, idiopathic pancreatitis, type 3c diabetes mellitus, latestage pancreatic cancer, and cancer cachexia.
 4. The method according toclaim 1, wherein the individual has an increased level of circulatingNMU relative to a reference level.
 5. The method according to claim 1,wherein the anti-NMU antibody or antigen binding region thereof ishumanized.
 6. The method according to claim 1, wherein the individual isobese and/or has a family history that includes diabetics.
 7. The methodaccording to claim 1, wherein: (a) the method is a therapeutic method,(b) the individual has a disease and is in need of increased circulatinginsulin levels, and (c) said dose is a therapeutically effective dosethat palliates, ameliorates, stabilizes, reverses, slows or delaysprogression of the disease, wherein the disease is selected from:diabetes, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC),pancreatitis, cystic fibrosis, and cancer cachexia.
 8. A method ofincreasing circulating insulin in an individual in need thereof, themethod comprising: administering to the individual, an anti-NMU antibodyor antigen binding region thereof that reduces binding between NMU andNMUR1, at a dose effective to increase the amount of circulating insulinin the individual, wherein the anti-NMU antibody or antigen bindingregion thereof comprises: (i) a light chain comprising a CDR-L1, CDR-L2,and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs:18-20, respectively, and a heavy chain comprising a CDR-H1, CDR-H2, andCDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs:26-28; or (ii) a light chain comprising a CDR-L1, CDR-L2, and CDR-L3comprising the amino acid sequences set forth in SEQ ID NOs: 2-4,respectively, and a heavy chain comprising a CDR-H1, CDR-H2, and CDR-H3comprising the amino acid sequences set forth in SEQ ID NOs: 10-12. 9.The method according to claim 8, wherein: (a) the method is atherapeutic method, (b) the individual has a disease and is in need ofincreased circulating insulin levels, and (c) said dose is atherapeutically effective dose that palliates, ameliorates, stabilizes,reverses, slows or delays progression of the disease, wherein thedisease is selected from: diabetes, pancreatic cancer, pancreatic ductaladenocarcinoma (PDAC), pancreatitis, cystic fibrosis, and cancercachexia.
 10. The method according to claim 8, wherein the individual ischaracterized by at least one of the following: (i) the individual isobese and/or has a family history that includes diabetics; (ii) theindividual has diabetes or is suspected of having an increased risk ofdeveloping diabetes; (iii) the individual has a disease selected from:cystic fibrosis, familial pancreatitis, idiopathic pancreatitis, type 3cdiabetes mellitus, late stage pancreatic cancer, and cancer cachexia.11. The method according to claim 8, wherein the anti-NMU antibody orantigen binding region thereof is humanized.
 12. A method of increasingcirculating insulin in an individual in need thereof, the methodcomprising: administering to the individual, an anti-NMU antibody orantigen binding region thereof that reduces binding between NMU andNMUR1, at a dose effective to increase the amount of circulating insulinin the individual, wherein the anti-NMU antibody or antigen bindingregion thereof comprises a heavy chain comprising a CDR-H1, CDR-H2, andCDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs:26-28, respectively, or SEQ ID NOs: 10-12, respectively.
 13. The methodaccording to claim 12, wherein: (a) the method is a therapeutic method,(b) the individual has a disease and is in need of increased circulatinginsulin levels, and (c) said dose is a therapeutically effective dosethat palliates, ameliorates, stabilizes, reverses, slows or delaysprogression of the disease, wherein the disease is selected from:diabetes, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC),pancreatitis, cystic fibrosis, and cancer cachexia.
 14. The methodaccording to claim 12, wherein the individual is characterized by atleast one of the following: (i) the individual is obese and/or has afamily history that includes diabetics; (ii) the individual has diabetesor is suspected of having an increased risk of developing diabetes;(iii) the individual has a disease selected from: cystic fibrosis,familial pancreatitis, idiopathic pancreatitis, type 3c diabetesmellitus, late stage pancreatic cancer, and cancer cachexia.
 15. Themethod according to claim 12, wherein the anti-NMU antibody or antigenbinding region thereof is humanized.
 16. A method of increasingcirculating insulin in an individual in need thereof, the methodcomprising: administering to the individual, an anti-NMU antibody orantigen binding region thereof that reduces binding between NMU andNMUR1, at a dose effective to increase the amount of circulating insulinin the individual, wherein the anti-NMU antibody or antigen bindingregion thereof comprises a first CDR comprising the amino acid sequenceset forth in SEQ ID NO: 18, a second CDR comprising the amino acidsequence set forth in SEQ ID NO: 19, a third CDR comprising the aminoacid sequence set forth in SEQ ID NO: 20, a fourth CDR comprising theamino acid sequence set forth in SEQ ID NO: 26, a fifth CDR comprisingthe amino acid sequence set forth in SEQ ID NO: 27, and a sixth CDRcomprising the amino acid sequence set forth in SEQ ID NO:
 28. 17. Themethod according to claim 16, wherein: (a) the method is a therapeuticmethod, (b) the individual has a disease and is in need of increasedcirculating insulin levels, and (c) said dose is a therapeuticallyeffective dose that palliates, ameliorates, stabilizes, reverses, slowsor delays progression of the disease, wherein the disease is selectedfrom: diabetes, pancreatic cancer, pancreatic ductal adenocarcinoma(PDAC), pancreatitis, cystic fibrosis, and cancer cachexia.
 18. Themethod according to claim 16, wherein the individual is characterized byat least one of the following: (i) the individual is obese and/or has afamily history that includes diabetics; (ii) the individual has diabetesor is suspected of having an increased risk of developing diabetes;(iii) the individual has a disease selected from: cystic fibrosis,familial pancreatitis, idiopathic pancreatitis, type 3c diabetesmellitus, late stage pancreatic cancer, and cancer cachexia.
 19. Themethod according to claim 16, wherein the anti-NMU antibody or antigenbinding region thereof is humanized.
 20. A method of increasingcirculating insulin in an individual in need thereof, the methodcomprising: administering to the individual, an anti-NMU antibody orantigen binding region thereof that reduces binding between NMU andNMUR1, at a dose effective to increase the amount of circulating insulinin the individual, wherein the anti-NMU antibody or antigen bindingregion thereof comprises a first CDR comprising the amino acid sequenceset forth in SEQ ID NO: 2, a second CDR comprising the amino acidsequence set forth in SEQ ID NO: 3, a third CDR comprising the aminoacid sequence set forth in SEQ ID NO: 4, a fourth CDR comprising theamino acid sequence set forth in SEQ ID NO: 10, a fifth CDR comprisingthe amino acid sequence set forth in SEQ ID NO: 11, and a sixth CDRcomprising the amino acid sequence set forth in SEQ ID NO:
 12. 21. Themethod according to claim 20, wherein: (a) the method is a therapeuticmethod, (b) the individual has a disease and is in need of increasedcirculating insulin levels, and (c) said dose is a therapeuticallyeffective dose that palliates, ameliorates, stabilizes, reverses, slowsor delays progression of the disease, wherein the disease is selectedfrom: diabetes, pancreatic cancer, pancreatic ductal adenocarcinoma(PDAC), pancreatitis, cystic fibrosis, and cancer cachexia.
 22. Themethod according to claim 20, wherein the individual is characterized byat least one of the following: (i) the individual is obese and/or has afamily history that includes diabetics; (ii) the individual has diabetesor is suspected of having an increased risk of developing diabetes;(iii) the individual has a disease selected from: cystic fibrosis,familial pancreatitis, idiopathic pancreatitis, type 3c diabetesmellitus, late stage pancreatic cancer, and cancer cachexia.
 23. Themethod according to claim 20, wherein the anti-NMU antibody or antigenbinding region thereof is humanized.